Non-hemolytic LLO fusion proteins and methods of utilizing same

ABSTRACT

The present invention provides recombinant proteins or peptides comprising a mutated listeriolysin O (LLO) protein or fragment thereof, comprising a substitution or internal deletion of the cholesterol-binding domain or a portion thereof, fusion proteins or peptides comprising same, nucleotide molecules encoding same, and vaccine vectors comprising or encoding same. The present invention also provides methods of utilizing recombinant proteins, peptides, nucleotide molecules, and vaccine vectors of the present invention to induce an immune response to a peptide of interest.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. application Ser.No. 14/163,269, filed Feb. 12, 2014, which is a Divisional of U.S.application Ser. No. 12/213,696 filed Jun. 23, 2008. These applicationsare hereby incorporated in their entirety by reference herein.

GOVERNMENT INTEREST STATEMENT

This invention was made in whole or in part with government supportunder Grant Number R43 CA108129-01 (SBIR), awarded by the NationalInstitutes of Health. The government may have certain rights in theinvention.

FIELD OF INVENTION

The present invention provides recombinant proteins or peptidescomprising a mutated listeriolysin O (LLO) protein or fragment thereof,comprising a substitution or internal deletion of thecholesterol-binding domain or a portion thereof, fusion proteins orpeptides comprising same, nucleotide molecules encoding same, andvaccine vectors comprising or encoding same. The present invention alsoprovides methods of utilizing recombinant proteins, peptides, nucleotidemolecules, and vaccine vectors of the present invention to induce animmune response to a peptide of interest.

BACKGROUND OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. Bacterialantigens such as Salmonella enterica and Mycobacterium bovis BCG remainin the phagosome and stimulate CD4⁺ T-cells via antigen presentationthrough major histocompatibility class II molecules. In contrast,bacterial antigens such as Listeria monocytogenes exit the phagosomeinto the cytoplasm. The phagolysosomal escape of L. monocytogenes is aunique mechanism, which facilitates major histocompatibility class Iantigen presentation of Listerial antigens. This escape is dependentupon the pore-forming sulfhydryl-activated cytolysin, listeriolysin O(LLO).

There exists a long-felt need to develop compositions and methods toenhance the immunogenicity of antigens, especially antigens useful inthe prevention and treatment of tumors and intracellular pathogens.

SUMMARY OF THE INVENTION

The present invention provides a recombinant protein comprising amutated listeriolysin O (LLO) protein or fragment thereof, containing amutation in or a substitution or internal deletion of thecholesterol-binding domain, fusion proteins or peptides comprising same,nucleotide molecules encoding same, and vaccine vectors comprising orencoding same. The present invention also provides methods of utilizingrecombinant proteins, peptides, nucleotide molecules, and vaccinevectors of the present invention to induce an immune response to apeptide of interest.

The present invention provides a recombinant protein comprising alisteriolysin O (LLO) protein or N-terminal fragment thereof, whereinsaid LLO protein or N-terminal fragment comprises a mutation in acholesterol-binding domain (CBD), wherein said mutation comprises asubstitution of a 1-50 amino acid peptide comprising a CBD as set forthin SEQ ID NO: 18 for a 1-50 amino acid non-LLO peptide, wherein saidrecombinant protein exhibits a greater than 100-fold reduction inhemolytic activity relative to wild-type LLO.

In another embodiment, the present invention provides a recombinantprotein comprising a listeriolysin O (LLO) protein or N-terminalfragment thereof, wherein said LLO protein or N-terminal fragmentcomprises a mutation in a cholesterol-binding domain (CBD), wherein saidmutation comprises a substitution of residue C484, W491, W492, of SEQ IDNO: 37 or a combination thereof, wherein said recombinant proteinexhibits a greater than 100-fold reduction in hemolytic activityrelative to wild-type LLO.

In another embodiment, the present invention provides a recombinantprotein comprising (a) a listeriolysin O (LLO) protein or N-terminalfragment thereof, wherein said LLO protein or N-terminal fragmentthereof comprises a 1-50 amino acid internal deletion in thecholesterol-binding domain of the LLO protein as set forth in SEQ ID NO:18; and (b) a heterologous peptide of interest, wherein said recombinantprotein exhibits a greater than 100-fold reduction in hemolytic activityrelative to wild-type LLO.

In one embodiment, the mutated LLO protein or mutated N-terminal LLOprotein fragment comprises a deletion of the signal peptide sequencethereof. In another embodiment, the mutated LLO protein or mutatedN-terminal LLO fragment comprises the signal peptide sequence thereof.In another embodiment, the recombinant protein comprises a heterologouspeptide of interest. In another embodiment, the recombinant proteincomprises a non-LLO peptide, which in one embodiment, comprises saidheterologous peptide of interest. In another embodiment, theheterologous peptide of interest is a full-length protein, which in oneembodiment, comprises an antigenic peptide. In one embodiment, theprotein is an NY-ESO-1 protein. In another embodiment, the protein is aHuman Papilloma Virus (HPV) E7 protein. In another embodiment, theprotein is a B-cell receptor (BCR) protein. In another embodiment, theheterologous peptide of interest is an antigenic peptide. In anotherembodiment, the antigenic peptide is an NY-ESO-1 peptide. In anotherembodiment, the antigenic peptide is a Human Papilloma Virus (HPV) E7peptide. In another embodiment, the antigenic peptide is a B-cellreceptor (BCR) peptide. In another embodiment, the antigenic peptide isa wherein said antigenic peptide is a Human Papilloma Virus (HPV)-16-E6,HPV-16-E7, HPV-18-E6, HPV-18-E7, a Her/2-neu antigen, a ProstateSpecific Antigen (PSA), Prostate Stem Cell Antigen (PSCA), a StratumCorneum Chymotryptic Enzyme (SCCE) antigen, Wilms tumor antigen 1(WT-1), human telomerase reverse transcriptase (hTERT), Proteinase 3,Tyrosinase Related Protein 2 (TRP2), High Molecular Weight MelanomaAssociated Antigen (HMW-MAA), synovial sarcoma, X (SSX)-2,carcinoembryonic antigen (CEA), MAGE-A, interleukin-13 Receptor alpha(IL13-R alpha), Carbonic anhydrase IX (CAIX), survivin, GP100, orTestisin peptide.

In another embodiment, the present invention provides a vaccinecomprising the recombinant protein and an adjuvant. In anotherembodiment, the adjuvant comprises a granulocyte/macrophagecolony-stimulating factor (GM-CSF) protein, a nucleotide moleculeencoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or anunmethylated CpG-containing oligonucleotide. In another embodiment, thepresent invention provides a composition comprising the recombinantprotein and a heterologous peptide of interest, wherein said recombinantprotein is not covalently bound to said heterologous peptide ofinterest. In another embodiment, the present invention provides avaccine comprising such a composition and an adjuvant. In anotherembodiment, the present invention provides a recombinant vaccine vectorencoding the recombinant protein. In another embodiment, the presentinvention provides a nucleotide molecule encoding the recombinantprotein.

In another embodiment, the present invention provides a vaccinecomprising the nucleotide molecule. In another embodiment, the presentinvention provides a recombinant Listeria strain comprising therecombinant protein or peptide. In another embodiment, the presentinvention provides a method for inducing an immune response in asubject, comprising administering to said subject the recombinantprotein or peptide. In another embodiment, the present inventionprovides a method for inducing an immune response in a subject,comprising administering to said subject the composition. In anotherembodiment, the present invention provides a method for inducing animmune response in a subject, comprising administering to said subjectthe recombinant vaccine vector wherein said non-LLO protein or peptideof said recombinant protein or peptide comprises an antigenic peptide ofinterest, thereby inducing an immune response against said antigenicpeptide of interest. In another embodiment, the present inventionprovides a method for inducing an immune response in a subject,comprising administering to said subject the recombinant vaccine vectorthat further comprises a heterologous peptide of interest, therebyinducing an immune response against said heterologous peptide ofinterest. In another embodiment, the present invention provides a methodfor inducing an immune response in a subject, comprising administeringto said subject a recombinant Listeria strain wherein said non-LLOprotein or peptide of said recombinant protein comprises an antigenicpeptide of interest, thereby inducing an immune response against saidantigenic peptide of interest. In another embodiment, the presentinvention provides a method for inducing an immune response in asubject, comprising administering to said subject the recombinantListeria strain and a vector encoding a heterologous peptide of interestthereby inducing an immune response against said heterologous peptide ofinterest.

In another embodiment, the present invention provides a method forinducing an immune response in a subject against an NY-ESO-1-expressingcancer cell selected from an ovarian melanoma cell and a lung cancercell, the method comprising the step of administering to said subject arecombinant protein of the present invention. In another embodiment, thepresent invention provides a method for treating, inhibiting, orsuppressing an NY-ESO-1-expressing tumor selected from an ovarianmelanoma tumor and a lung cancer tumor in a subject, the methodcomprising the step of administering to said subject the recombinantprotein of the present invention.

In another embodiment, the present invention provides a method forinducing an immune response in a subject against an HPV E7-expressingcancer cell selected from a cervical cancer cell and a head-and-neckcancer cell, the method comprising the step of administering to saidsubject the recombinant protein of the present invention. In anotherembodiment, the present invention provides a method for treating,inhibiting, or suppressing an HPV E7-expressing tumor selected from acervical cancer tumor and a head-and-neck cancer tumor in a subject, themethod comprising the step of administering to said subject therecombinant protein of the present invention. In another embodiment, thepresent invention provides a method for inducing an immune response in asubject against a B-cell receptor (BCR)-expressing lymphoma, the methodcomprising the step of administering to said subject the recombinantprotein of the present invention. In another embodiment, the presentinvention provides a method for treating, inhibiting, or suppressing aB-cell receptor (BCR)-expressing lymphoma in a subject, the methodcomprising the step of administering to said subject the recombinantprotein of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Lm-E7 and Lm-LLO-E7 use different expression systems to expressand secrete E7. Lm-E7 was generated by introducing a gene cassette intothe orfZ domain of the L. monocytogenes genome (A). The hly promoterdrives expression of the hly signal sequence and the first five aminoacids (AA) of LLO followed by HPV-16 E7. B), Lm-LLO-E7 was generated bytransforming the prfA-strain XFL-7 with the plasmid pGG-55. pGG-55 hasthe hly promoter driving expression of a nonhemolytic fusion of LLO-E7.pGG-55 also contains the prfA gene to select for retention of theplasmid by XFL-7 in vivo.

FIG. 2. Lm-E7 and Lm-LLO-E7 secrete E7. Lm-Gag (lane 1), Lm-E7 (lane 2),Lm-LLO-NP (lane 3), Lm-LLO-E7 (lane 4), XFL-7 (lane 5), and 10403S (lane6) were grown overnight at 37° C. in Luria-Bertoni broth. Equivalentnumbers of bacteria, as determined by OD at 600 nm absorbance, werepelleted and 18 ml of each supernatant was TCA precipitated. E7expression was analyzed by Western blot. The blot was probed with ananti-E7 mAb, followed by HRP-conjugated anti-mouse (Amersham), thendeveloped using ECL detection reagents.

FIG. 3 A-B. FIG. 3A. Tumor immunotherapeutic efficacy of LLO-E7 fusions.Tumor size in millimeters in mice is shown at 7, 14, 21, 28 and 56 dayspost tumor-inoculation. Naive mice: open-circles; Lm-LLO-E7: filledcircles; Lm-E7: squares; Lm-Gag: open diamonds; and Lm-LLO-NP: filledtriangles. FIG. 3B. Tumor immunotherapeutic efficacy of LLO-Ova fusions.

FIG. 4. Splenocytes from Lm-LLO-E7-immunized mice proliferate whenexposed to TC-1 cells. C57BL/6 mice were immunized and boosted withLm-LLO-E7, Lm-E7, or control rLm strains. Splenocytes were harvested 6days after the boost and plated with irradiated TC-1 cells at the ratiosshown. The cells were pulsed with ³H thymidine and harvested. Cpm isdefined as (experimental cpm)—(no-TC-1 control).

FIG. 5. Tumor immunotherapeutic efficacy of NP antigen expressed in LM.Tumor size in millimeters in mice is shown at 10, 17, 24, and 38 dayspost tumor-inoculation. Naive mice: X's; mice administered Lm-LLO-NP:filled diamonds; Lm-NP: squares; Lm-Gag: open circles.

FIG. 6. Depiction of vaccinia virus constructs expressing differentforms of HPV16 E7 protein.

FIG. 7. Vac-LLO-E7 causes long-term regression of tumors establishedfrom 2×10⁵ TC-1 cells injected s.c. into C57BL/6 mice. Mice wereinjected 11 and 18 days after tumor challenge with 10⁷ PFU ofVac-LLO-E7, Vac-SigE7LAMP-1, or VacE7/mouse i.p. or were untreated(naive). 8 mice per treatment group were used, and the cross section foreach tumor (average of 2 measurements) is shown for the indicated daysafter tumor inoculation.

FIG. 8A-B. SOE mutagenesis strategy. Decreasing/lowering the virulenceof LLO was achieved by mutating the 4th domain of LLO. This domaincontains a cholesterol binding site allowing it to bind to membraneswhere it oligomerizes to form pores. FIG. 8C. Shows fragments of fulllength LLO (rLLO529). Recombinant LLO, rLLO493, represents a LLON-terminal fragment spanning from amino acids 1-493 (including thesignal sequence). Recombinant LLO, rLLO482, represents an N-terminal LLOfragment (including a deletion of the cholesterol binding domain—aminoacids 483-493-) spanning from amino acids 1-482 (including the signalsequence). Recombinant LLO, rLLO415, represents a N-terminal LLOfragment (including a deletion of the cholesterol binding domain-aminoacids 483-493-) spanning from amino acids 1-415 (including the signalsequence). Recombinant LLO, rLLO59-415, represents a N-terminal LLOfragment that spans from amino acids 59-415 (excluding the cholesterolbinding domain). Recombinant LLO, rLLO416-529, represents a N-terminalLLO fragment that spans from amino acids 416-529 and includes thecholesterol binding domain.

FIG. 9 A-B. Expression of mutant LLO proteins by Coomassie staining(FIG. 9A) and Western blot (FIG. 9B).

FIG. 10 A-B. Hemolytic activity of mutant LLO (mutLLO and ctLLO)proteins at pH 5.5 (FIG. 10A) and 7.4 (FIG. 10B).

FIG. 11. Expression of 38C13 soluble protein yields 2.34 mg solubleprotein from the cell pellet per liter of induction medium. Induction of38C13scFv protein expression in BL21* was performed using 1 mM IPTG inSuperbroth containing 0.5% glycine and 1% triton X-100 at 20° C. for 16hours. Soluble proteins were extracted from the cell pellet using aprotocol including freeze/thaw in nonionic detergent, lysozyme andsonication. 38scFv proteins were purified from the extracted solubleproteins in the anti-idiotype sepharose column Samples from the affinitychromatography study were electophoresed on SDS-PAGE gels and Coumassiestaining (A) or myc tag Western (B). The flow through (ft) and washfractions contained the 38scFv protein, indicating the Id-Sepharose®column was overloaded with the protein. These fractions were re-loadedonto the Id-Sepharose® slurry and further recombinant protein recovered.Lanes: 1—M Wt; 2—soluble fraction; 3—ft; 4—wash at 1 ml; 5—wash at 100ml; 6—pooled elution fraction.

FIG. 12. Strategy for 38scFv protein expression in E. coli andsubsequent purification by affinity chromatography. Diagram shows thepathway for production of purified 38C13scFv and subsequent purificationon an immunoaffinity column with the anti-Id antibody S1C5.

FIG. 13. ELISA assay to quantitate 38C13scFv production in inductioncultures, to test correct folding of the protein after conjugation toimmunogens, and to monitor the humoral immune response. The principle ofthe ELISA assay is depicted in (a). A standard curve (b) shows thechange in A(405-490) for serial dilutions of purified 38scFv.

FIG. 14. Production of whole 38Id protein-LLO conjugates for vaccinestudies. The 38C13 IgM protein was secreted by the 38C13A1.2 hybridomainto the bioreactor culture supernatant. The 38C13 IgM protein waspurified from the culture supernatant using differential ammoniumsulfate precipitation. Soluble LLO-His protein was expressed in E. colifollowing induction by IPTG, the soluble protein was then purified on aNi+-NTA column and purity confirmed by Coumassie and Western blot usingthe LLO antibody B3-19. The 38C13 Id protein was conjugated toglutaraldehyde, dialyzed against PBS and passed through a Polymixin Bcolumn to remove endotoxin; endotoxin removal was confirmed by the LALassay. The hemolytic activity of the 38Id-LLO conjugate was then testedusing sheep red cells and compared to purified LLO, the 38Id-LLO wasfound to be non-hemolytic.

FIG. 15. Samples from differential ammonium sulfate precipitation ofbioreactor supernatant following culture of the hybridoma 38C13A1.2 wererun by SDS-PAGE gel and stained by Coumassie. The 38C13 idiotype proteinwas recovered from the 45% fraction and characterized in both reducingand non-reducing conditions.

FIG. 16. Soluble proteins were recovered from E. coli strain BL21*following an induction expression culture in LB medium and 1 mM IPTG for18 hours at 30 C. Recombinant LLO-His was then purified on a Ni+-NTAcolumn; the purity of the elution fractions were confirmed by SDS PAGEfollowed by a Coumassie stain or a Western blot performed using MabB3-19.

FIG. 17. 38C13 idiotype (Id) protein was conjugated to either KLH (leftpanel) or LLO (right panel). Conjugation of 38C13 idiotype protein toKLH or LLO is complete, as confirmed by Coumassie stain on SDS-PAGE gelrun under reducing and non-reducing conditions; both 38Id-KLH and38Id-LLO conjugates show no evidence of free 38Id or the immunogenicproteins.

FIG. 18. Principle of the assay system designed to demonstrate thepresence of the 38C13 idiotype epitope. The presence of the 38C13idiotype epitope was confirmed using a blocking assay, in this systemthe anti-38C13 idiotype antibody 51C5-FITC is incubated with the Idprotein or the conjugates 38Id-KLH or 38Id-LLO. Subsequently the bindingof the S1C5-FITC to the 38C13 cell line B-cell receptor (BCR) isassessed by flow cytometry. In the presence of 38Id protein, the bindingof S1C5-FITC to 38C13 lymphoma is impaired.

FIG. 19. 38C13 Id protein conjugated to LLO or KLH retains the bindingsite for the S1C5 MAb and inhibits binding of 51C5-FITC to 38C13lymphoma cells. Arrow marks approximately 5-fold reduction influorescence. For unconjugated protein (top left panel), thiscorresponded to 100 ng protein. For 38Id-KLH, this corresponded to 10mcg protein (upper right panel). For 38Id-LLO, this corresponded tobetween 100 ng-1 mcg protein (lower right panel).

FIG. 20 A-B. Id-LLO immunization protects mice from 38C13 lymphomachallenge. Mice were immunized with 38Id or 38Id conjugates andchallenged with 38C13 lymphoma (FIG. 20A). The development of s.c.lymphoma was monitored for each mouse over the next 60 days (FIG. 20B),and the results presented as the frequency of each vaccine group tumorfree. Statistical analysis was performed (non-parametric Kaplan-Meier,Log Rank Mantel-Cox test) using SPSS software. Asterisk-result isstatistically different (p<0.05) from control groups.

FIG. 21 A-B. Id protein vaccine induces anti-idiotype antibodies whenthe Id protein is conjugated to KLH or LLO. Peripheral blood sampleswere collected from individual mice prior to and 12 days after eachimmunization. The serum samples were then tested by ELISA assay for thepresence of anti-idiotype antibodies. The results for each vaccine grouphave been summarized in. Mice from the 38Id-LLO and 38Id-KLH vaccinegroups were the only vaccine groups with sera positive for anti-idiotypeantibodies (FIG. 21A). An isotyping assay was performed to characterizethe anti-idiotype antibodies induced by 38Id-LLO versus 38Id-KLH.Following a single immunization with 38Id-LLO, a high titer polyclonalresponse was induced with equivalent levels of IgG1 and IgG2aanti-idioype antibodies (FIG. 21B). The level of the 38Id-LLO inducedantibodies increased after the second immunization; however the ratio ofIgG1:IgG2a (1.0) remained the same. In contrast, the 38Id-KLH vaccineinduced a higher level of IgG1 versus IgG2a anti-idiotype antibodiesafter both immunizations (IgG1:IgG2a ratio was 1.8 and 1.3 respectively(FIG. 21B).

FIG. 22. Anti-idiotype antibodies are present in mouse serum afterimmunization and tumor challenges. To confirm the above results, theability of immunized mouse serum to block binding of S1C5-FITC to 38C13cells was measured, as a decrease in fluorescence by FACS. In the firstexperiment (A), the binding specificity of S1C5 to the 38C13 lymphomaidiotype was verified. Subsequently, the inhibition of S1C5 binding to38C13 cells by mouse serum (taken at various stages through Id-LLOimmunization and after tumor challenges) was investigated (B).

FIG. 23 A-D. Id-LLO immunization induces a Th1 response andantigen-specific CD8 T cells. Cells were harvested from DLN 14 daysafter s.c. immunization. CFSE-stained DLN cells were incubated withpurified proteins for 5 days before being re-stimulated withPMA/Ionomycin for 5 hours in the presence of monensin. Cells werestained for surface CD4 and CD8, and then fixed and stained forintracellular cytokine. Percentage (mean±SD) of gated cells secretingcytokines is depicted. (FIG. 23A) CD4 T cell IFN-γ secretion; (FIG. 23B)CD4 T cell IL-4 secretion; (FIG. 23C) CD8 T cell IFN-γ secretion; (FIG.23D) CD4 proliferation results. Student t-test was used to analyze thedata; asterisk indicates a significantly different result with in vitroprotein stimulation (p=<0.05) compared to media only in that vaccinegroup.

FIG. 24. Representative dot plots of CD4 CFSE proliferation assay. whichFIG. 23D data were calculated. DLNs were collected 14 days after s.c.immunization and cells harvested. CFSE-stained DLN cells were thenincubated with purified proteins for 5 days before being re-stimulatedwith PMA/Ionomycin for 5 hours in the presence of monensin. Cells werethen stained for surface CD4 and CD8, fixed and stained forintracellular cytokine. Data was acquired on a FACSCalibur and analyzedby FlowJo software. Representative dotplots for CD4 CFSE proliferationare shown in FIG. 24.

FIG. 25 A-C. Co-inoculation of post-Id-LLO serum, CD4 or CD8 T cellsinhibits the growth of 38C13 lymphoma cells. Transfer of serum, CD4 orCD8 T cells after Id-LLO immunization protects from in vivo challengewith 38C13 lymphoma. Experimental design is depicted in (FIG. 25A). Micewere immunized with 2 rounds of Id-LLO+mGMCSF; control group was naïvemice. Fourteen days after the second round of immunization, DLNs werecollected and purified DLN CD4 or CD8 T cells were prepared as well as apool of serum. The serum, CD4 or CD8 T cells were then co-inoculateds.c. with 38C13 cells on the left flank into recipient mice (8 pergroup), and mice were monitored for 60 days to assess lymphomadevelopment. Results for serum transfer are shown in (FIG. 25B), CD4 orCD8 transfer in (FIG. 25C). Statistical analysis was performed(non-parametric Kaplan-Meier, Log Rank Mantel-Cox test) using SPSSsoftware). Asterisk-statistical difference (p<0.05) in anti-tumorefficacy between effectors from immunized and naïve mice.

FIG. 26. Mice immunized with 38Id-KLH or 38Id-LLO are protected from38C13 challenge on the opposite flank to the initial immunization andchallenge.

FIG. 27. The ability of rLLO+rE7 chemically conjugated and rLLO+rE7mixed together to impact on TC-1 growth.

FIG. 28. The ability of rE7 and rLLO protein to impact on TC-1 growth.

FIG. 29. The ability of recombinant detoxified LLOE7 (rDTLLO-E7; wholesequence) and rDTLLO-E7 (chimera) to impact on TC-1 growth.

FIG. 30. TC-1 tumor regression after immunization with rE7, rLLO,rLLO+E7 and rDTLLO-E7.

FIG. 31. TC-1 tumor regression after immunization with rDTLLO-chimera.

FIG. 32. TC-1 tumor regression after immunization with rE7, rDTLLO,rDTLLO+rE7, rDTLLO-E7 and rDT-LLO-E7-chimera.

FIG. 33. TC-1 tumor regression immunized with ActA-E7 and E7 protein.

FIG. 34. TC-1 tumor regression immunized with ActA+E7 and E7 protein.

FIG. 35. TC-1 tumor regression immunized with ActA and E7 protein.

FIG. 36. TC-1 tumor regression after immunization with rDTLLO-chimera

FIG. 37A. DetoxLLO Induces Cytokine mRNA expression by Bone Marrow (BM)Macrophages. 8e5 Day 7 BMDCs were thawed overnight at 37° C. in RF10media. Next, BMDCs were centrifugated and resuspended in 1 mL of freshRF10 at 37° C. for 1 hr. BMDCs were treated w/ 40 mcg/mL of LLOE7 andmolar equivalents of E7 and LLO (or with PBS as negative control or 1mcg/mL LPS as positive control). After 2 and 24 hrs, cells werecollected by centrifugation and media saved for ELISA. RNA was extractedfrom cells and converted to cDNA. cDNA was then subjected to qPCRanalysis with primers for various cytokines. This figure shows inductionof TNF-α after 2 hours.

FIG. 37B shows induction of TNF-α after 24 hrs.

FIG. 37C shows induction of IL-12 after 2 hours.

FIG. 37D shows induction of IL-12 after 24 hrs.

FIG. 37E shows induction of ISG15 after 24 hours.

FIG. 38A. Detox LLO Induces Cytokine Secretion by BM Macrophages. Sametreatment protocol as described for FIG. 37, except media was subjectedto ELISA analysis after treatments. This figure shows induction of TNF-αafter 2 hours and 24 hours.

FIG. 38B. Detox LLO Induces Cytokine Secretion by BM Macrophages. Sametreatment protocol as described for FIG. 37, except media was subjectedto ELISA analysis after treatments. This figure shows induction of IL-12after 2 hours and 24 hours.

FIG. 39A. Detox LLO Upregulates DC Maturation Markers. Bone marrow wascollected from the femurs of C57BL/6 mice at 6-8 wk of age. After 7 daysof culture, nonadherent cells were collected, washed, and plated at2×10^6/ml and then pulsed with either E7 (10 mcg/ml), LLO (40 mcg/ml),or LLOE7 (50 mcg/ml) plus LLO (40 mcg/ml) for 16 hr in 37° C., 5% CO₂.Cells were stained with APC-labeled mAbs specific for mouse CD11c, orFITC-labeled mAb specific for mouse CD86, MHC class II, CD40.Isotype-matched mouse IgG was used as a negative control and subtractedfrom the background. Cells were incubated with mAbs for 30 min at 4° C.in the dark. Following two washes with PBS, 10 μl of 7AAD (BeckmanCoulter, Marseille, France) was added 10 min before cells were analyzedon a FACS flow cytometer. The live cell population is shown aspercentage of CD11c positive cells. This figure shows upregulation ofCD86 as compared to controls.

FIG. 39B shows upregulation of CD40 as compared to controls.

FIG. 39C shows upregulation of MHCII as compared to controls.

FIG. 40. Regression of TC-1 Tumors by LLO-fused E7. 2×10^5 TC-1 tumorcells were established s.c in 8 mice per vaccine group. Mice wereimmunized s.c. with 50 μg of E7, 200 μg of LLO, 250 μg of LLOE7, or 50μg of E7 plus 200 μg of LLO on Days 3 and 10.

FIG. 41. Nuclear translocation of NFkappaB after stimulation withDt.LLO. J774 macrophage cell line used as model system for antigenpresenting cells (APCs). 5×10^5 cells per well (6 well dish) were platedin a total volume 1 ml. Cells were stained with anti-NF-κB (P65)—FITC(green fluorescence) and DAPI for nucleus (blue fluorescence). In B, D,and F, cells were also stained after 24 hours with anti-CD11B-PE(M1/170, eBioscence). The fluorescent micrograph is shown at 40×magnification. NF-kappaB is located in the cytoplasm after treatment ofcells with media alone (no activation) (A). Media-treated cellsdemonstrate weak Cd11b staining (B). After overnight (24 hr) stimulationwith Dt.LLO (30 mcg), NFkappaB moved out of the cytoplasm into thenucleus (C) and there is an increase in CD11b staining (D). Similarly,after overnight stimulation (24 hr) with LPS (10 mcg/ml, positivecontrol), NFkappaB was translocated to the nucleus (E), which is morediscernible with the halo made by the increased CD11b+ staining of theplasma membrane (F).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides recombinant proteins and peptidescomprising a mutated listeriolysin O (LLO) protein or fragment thereof,comprising a substitution or internal deletion that includes thecholesterol-binding domain, fusion peptides comprising same, nucleotidemolecules encoding same, and vaccine vectors comprising or encodingsame. The present invention also provides methods of utilizingrecombinant peptides, nucleotide molecules, and vaccine vectors of thepresent invention to induce an immune response.

In one embodiment, the present invention provides a recombinant proteinor polypeptide comprising a listeriolysin O (LLO) protein, wherein saidLLO protein comprises a mutation of residues C484, W491, W492, or acombination thereof of the cholesterol-binding domain (CBD) of said LLOprotein. In one embodiment, said C484, W491, and W492 residues areresidues C484, W491, and W492 of SEQ ID NO: 37, while in anotherembodiment, they are corresponding residues as can be deduced usingsequence alignments, as is known to one of skill in the art. In oneembodiment, residues C484, W491, and W492 are mutated. In oneembodiment, a mutation is a substitution, in another embodiment, adeletion. In one embodiment, the entire CBD is mutated, while in anotherembodiment, portions of the CBD are mutated, while in anotherembodiment, only specific residues within the CBD are mutated.

In another embodiment, the LLO fragment is an N-terminal LLO fragment.In another embodiment, the LLO fragment is at least 492 amino acids (AA)long. In another embodiment, the LLO fragment is 492-528 AA long. Inanother embodiment, the non-LLO peptide is 1-50 amino acids long. Inanother embodiment, the mutated region is 1-50 amino acids long. Inanother embodiment, the non-LLO peptide is the same length as themutated region. In another embodiment, the non-LLO peptide is shorter,or in another embodiment, longer, than the mutated region. In anotherembodiment, the substitution is an inactivating mutation with respect tohemolytic activity. In another embodiment, the recombinant peptideexhibits a reduction in hemolytic activity relative to wild-type LLO. Inanother embodiment, the recombinant peptide is non-hemolytic. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, the present invention provides a recombinant proteinor polypeptide comprising a mutated LLO protein or fragment thereof,wherein the mutated LLO protein or fragment thereof contains asubstitution of a non-LLO peptide for a mutated region of the mutatedLLO protein or fragment thereof, the mutated region comprising a residueselected from C484, W491, and W492. In another embodiment, the LLOfragment is an N-terminal LLO fragment. In another embodiment, the LLOfragment is at least 492 amino acids (AA) long. In another embodiment,the LLO fragment is 492-528 AA long. In another embodiment, the non-LLOpeptide is 1-50 amino acids long. In another embodiment, the mutatedregion is 1-50 amino acids long. In another embodiment, the non-LLOpeptide is the same length as the mutated region. In another embodiment,the non-LLO peptide has a length different from the mutated region. Inanother embodiment, the substitution is an inactivating mutation withrespect to hemolytic activity. In another embodiment, the recombinantprotein or polypeptide exhibits a reduction in hemolytic activityrelative to wild-type LLO. In another embodiment, the recombinantprotein or polypeptide is non-hemolytic. Each possibility represents aseparate embodiment of the present invention.

As provided herein, a mutant LLO protein was created wherein residuesC484, W491, and W492 of LLO were substituted with alanine residues(Example 5). The mutated LLO protein, mutLLO, could be expressed andpurified in an E. coli expression system (Example 7) and exhibitedsubstantially reduced hemolytic activity relative to wild-type LLO(Example 8).

In another embodiment, the present invention provides a recombinantprotein or polypeptide comprising a mutated LLO protein or fragmentthereof, wherein the mutated LLO protein or fragment thereof contains asubstitution of a non-LLO peptide for a mutated region of the mutatedLLO protein or fragment thereof, the mutated region comprising thecholesterol-binding domain (CBD) of the mutated LLO protein or fragmentthereof. In another embodiment, the LLO fragment is an N-terminal LLOfragment. In another embodiment, the LLO fragment is at least 492 AAlong. In another embodiment, the LLO fragment is 492-528 AA long. Inanother embodiment, the non-LLO peptide is 1-50 amino acids long. Inanother embodiment, the mutated region is 11-50 amino acids long. Inanother embodiment, the non-LLO peptide is the same length as themutated region. In another embodiment, the non-LLO peptide has a lengthdifferent from the mutated region. In another embodiment, thesubstitution is an inactivating mutation with respect to hemolyticactivity. In another embodiment, the recombinant protein or polypeptideexhibits a reduction in hemolytic activity relative to wild-type LLO. Inanother embodiment, the recombinant protein or polypeptide isnon-hemolytic. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a recombinantprotein or polypeptide comprising a mutated LLO protein or fragmentthereof, wherein the mutated LLO protein or fragment thereof contains asubstitution of a non-LLO peptide for a mutated region of the mutatedLLO protein or fragment thereof, wherein the mutated region is afragment of the CBD of the mutated LLO protein or fragment thereof. Inanother embodiment, the LLO fragment is an N-terminal LLO fragment. Inanother embodiment, the LLO fragment is at least 492 AA long. In anotherembodiment, the LLO fragment is 492-528 AA long. In another embodiment,the non-LLO peptide is 1-50 amino acids long. In another embodiment, themutated region is 1-11 amino acids long. In another embodiment, thenon-LLO peptide is the same length as the mutated region. In anotherembodiment, the non-LLO peptide has a length different from the mutatedregion. In another embodiment, the substitution is an inactivatingmutation with respect to hemolytic activity. In another embodiment, therecombinant protein or polypeptide exhibits a reduction in hemolyticactivity relative to wild-type LLO. In another embodiment, therecombinant protein or polypeptide is non-hemolytic. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a recombinantprotein or polypeptide comprising a mutated LLO protein or fragmentthereof, wherein the mutated LLO protein or fragment thereof contains asubstitution of a 1-50 amino acid non-LLO peptide for a 1-50 amino acidmutated region of the mutated LLO protein or fragment thereof, whereinthe mutated region overlaps the CBD of the mutated LLO protein orfragment thereof. In another embodiment, the LLO fragment is anN-terminal LLO fragment. In another embodiment, the LLO fragment is atleast 492 AA long. In another embodiment, the LLO fragment is 492-528 AAlong. In another embodiment, the substitution is an inactivatingmutation with respect to hemolytic activity. In another embodiment, therecombinant protein or polypeptide exhibits a reduction in hemolyticactivity relative to wild-type LLO. In another embodiment, therecombinant protein or polypeptide is non-hemolytic. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a recombinantprotein or polypeptide comprising (a) a mutated LLO protein, wherein themutated LLO protein contains an internal deletion, the internal deletioncomprising the cholesterol-binding domain of the mutated LLO protein;and (b) a heterologous peptide of interest. In another embodiment, thesequence of the cholesterol-binding domain is set forth in SEQ ID NO:18. In another embodiment, the internal deletion is an 11-50 amino acidinternal deletion. In another embodiment, the internal deletion isinactivating with regard to the hemolytic activity of the recombinantprotein or polypeptide. In another embodiment, the recombinant proteinor polypeptide exhibits a reduction in hemolytic activity relative towild-type LLO. Each possibility represents another embodiment of thepresent invention.

In another embodiment, the present invention provides a recombinantprotein or polypeptide comprising (a) a mutated LLO protein, wherein themutated LLO protein contains an internal deletion, the internal deletioncomprising comprises a residue selected from C484, W491, and W492 of themutated LLO protein; and (b) a heterologous peptide of interest. Inanother embodiment, the internal deletion is a 1-50 amino acid internaldeletion. In another embodiment, the internal deletion is inactivatingwith regard to the hemolytic activity of the recombinant protein orpolypeptide. In another embodiment, the recombinant protein orpolypeptide exhibits a reduction in hemolytic activity relative towild-type LLO. Each possibility represents another embodiment of thepresent invention.

In another embodiment, the present invention provides a recombinantprotein or polypeptide comprising (a) a mutated LLO protein, wherein themutated LLO protein contains an internal deletion, the internal deletioncomprising a fragment of the cholesterol-binding domain of the mutatedLLO protein; and (b) a heterologous peptide of interest. In anotherembodiment, the internal deletion is a 1-11 amino acid internaldeletion. In another embodiment, the sequence of the cholesterol-bindingdomain is set forth in SEQ ID NO: 18. In another embodiment, theinternal deletion is inactivating with regard to the hemolytic activityof the recombinant protein or polypeptide. In another embodiment, therecombinant protein or polypeptide exhibits a reduction in hemolyticactivity relative to wild-type LLO. Each possibility represents anotherembodiment of the present invention.

In another embodiment, the present invention provides a vaccinecomprising an adjuvant, a recombinant protein or polypeptide of thepresent invention, and a heterologous peptide of interest. In anotherembodiment, the present invention provides a composition comprising anadjuvant, a recombinant protein or polypeptide of the present invention,and a heterologous antigenic peptide of interest. In another embodiment,the recombinant protein or polypeptide is not covalently bound to theheterologous peptide of interest. Each possibility represents a separateembodiment of the present invention.

The mutated region of methods and compositions of the present inventioncomprises, in another embodiment, residue C484 of SEQ ID NO: 37. Inanother embodiment, the mutated region comprises a correspondingcysteine residue of a homologous LLO protein. In another embodiment, themutated region comprises residue W491 of SEQ ID NO: 37. In anotherembodiment, the mutated region comprises a corresponding tryptophanresidue of a homologous LLO protein. In another embodiment, the mutatedregion comprises residue W492 of SEQ ID NO: 37. In another embodiment,the mutated region comprises a corresponding tryptophan residue of ahomologous LLO protein. Methods for identifying corresponding residuesof a homologous protein are well known in the art, and include, forexample, sequence alignment. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the mutated region comprises residues C484 andW491. In another embodiment, the mutated region comprises residues C484and W492. In another embodiment, the mutated region comprises residuesW491 and W492. In another embodiment, the mutated region comprisesresidues C484, W491, and W492. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the mutated region of methods and compositions ofthe present invention comprises the cholesterol-binding domain of themutated LLO protein or fragment thereof. For example, a mutated regionconsisting of residues 470-500, 470-510, or 480-500 of SEQ ID NO: 37comprises the CBD thereof (residues 483-493). In another embodiment, themutated region is a fragment of the CBD of the mutated LLO protein orfragment thereof. For example, as provided herein, residues C484, W491,and W492, each of which is a fragment of the CBD, were mutated toalanine residues (Example 5). Further, as provided herein, a fragment ofthe CBD, residues 484-492, was replaced with a heterologous sequencefrom NY-ESO-1 (Example 6). In another embodiment, the mutated regionoverlaps the CBD of the mutated LLO protein or fragment thereof. Forexample, a mutated region consisting of residues 470-490, 480-488,490-500, or 486-510 of SEQ ID NO: 37 comprises the CBD thereof. Inanother embodiment, a single peptide may have a deletion in the signalsequence and a mutation or substitution in the CBD. Each possibilityrepresents a separate embodiment of the present invention.

The length of the mutated region is, in another embodiment, 1-50 AA. Inanother embodiment, the length is 1-11 AA. In another embodiment, thelength is 2-11 AA. In another embodiment, the length is 3-11 AA. Inanother embodiment, the length is 4-11 AA. In another embodiment, thelength is 5-11 AA. In another embodiment, the length is 6-11 AA. Inanother embodiment, the length is 7-11 AA. In another embodiment, thelength is 8-11 AA. In another embodiment, the length is 9-11 AA. Inanother embodiment, the length is 10-11 AA. In another embodiment, thelength is 1-2 AA. In another embodiment, the length is 1-3 AA. Inanother embodiment, the length is 1-4 AA. In another embodiment, thelength is 1-5 AA. In another embodiment, the length is 1-6 AA. Inanother embodiment, the length is 1-7 AA. In another embodiment, thelength is 1-8 AA. In another embodiment, the length is 1-9 AA. Inanother embodiment, the length is 1-10 AA. In another embodiment, thelength is 2-3 AA. In another embodiment, the length is 2-4 AA. Inanother embodiment, the length is 2-5 AA. In another embodiment, thelength is 2-6 AA. In another embodiment, the length is 2-7 AA. Inanother embodiment, the length is 2-8 AA. In another embodiment, thelength is 2-9 AA. In another embodiment, the length is 2-10 AA. Inanother embodiment, the length is 3-4 AA. In another embodiment, thelength is 3-5 AA. In another embodiment, the length is 3-6 AA. Inanother embodiment, the length is 3-7 AA. In another embodiment, thelength is 3-8 AA. In another embodiment, the length is 3-9 AA. Inanother embodiment, the length is 3-10 AA. In another embodiment, thelength is 11-50 AA. In another embodiment, the length is 12-50 AA. Inanother embodiment, the length is 11-15 AA. In another embodiment, thelength is 11-20 AA. In another embodiment, the length is 11-25 AA. Inanother embodiment, the length is 11-30 AA. In another embodiment, thelength is 11-35 AA. In another embodiment, the length is 11-40 AA. Inanother embodiment, the length is 11-60 AA. In another embodiment, thelength is 11-70 AA. In another embodiment, the length is 11-80 AA. Inanother embodiment, the length is 11-90 AA. In another embodiment, thelength is 11-100 AA. In another embodiment, the length is 11-150 AA. Inanother embodiment, the length is 15-20 AA. In another embodiment, thelength is 15-25 AA. In another embodiment, the length is 15-30 AA. Inanother embodiment, the length is 15-35 AA. In another embodiment, thelength is 15-40 AA. In another embodiment, the length is 15-60 AA. Inanother embodiment, the length is 15-70 AA. In another embodiment, thelength is 15-80 AA. In another embodiment, the length is 15-90 AA. Inanother embodiment, the length is 15-100 AA. In another embodiment, thelength is 15-150 AA. In another embodiment, the length is 20-25 AA. Inanother embodiment, the length is 20-30 AA. In another embodiment, thelength is 20-35 AA. In another embodiment, the length is 20-40 AA. Inanother embodiment, the length is 20-60 AA. In another embodiment, thelength is 20-70 AA. In another embodiment, the length is 20-80 AA. Inanother embodiment, the length is 20-90 AA. In another embodiment, thelength is 20-100 AA. In another embodiment, the length is 20-150 AA. Inanother embodiment, the length is 30-35 AA. In another embodiment, thelength is 30-40 AA. In another embodiment, the length is 30-60 AA. Inanother embodiment, the length is 30-70 AA. In another embodiment, thelength is 30-80 AA. In another embodiment, the length is 30-90 AA. Inanother embodiment, the length is 30-100 AA. In another embodiment, thelength is 30-150 AA. Each possibility represents another embodiment ofthe present invention.

The substitution mutation of methods and compositions of the presentinvention is, in another embodiment, a mutation wherein the mutatedregion of the LLO protein or fragment thereof is replaced by an equalnumber of heterologous AA. In another embodiment, a larger number ofheterologous AA than the size of the mutated region is introduced. Inanother embodiment, a smaller number of heterologous AA than the size ofthe mutated region is introduced. Each possibility represents anotherembodiment of the present invention.

In another embodiment, the substitution mutation is a point mutation ofa single residue. In another embodiment, the substitution mutation is apoint mutation of 2 residues. In another embodiment, the substitutionmutation is a point mutation of 3 residues. In another embodiment, thesubstitution mutation is a point mutation of more than 3 residues. Inanother embodiment, the substitution mutation is a point mutation ofseveral residues. In another embodiment, the multiple residues includedin the point mutation are contiguous. In another embodiment, themultiple residues are not contiguous. Each possibility representsanother embodiment of the present invention.

The length of the non-LLO peptide that replaces the mutated region ofrecombinant protein or polypeptides of the present invention is, inanother embodiment, 1-50 AA. In another embodiment, the length is 1-11AA. In another embodiment, the length is 2-11 AA. In another embodiment,the length is 3-11 AA. In another embodiment, the length is 4-11 AA. Inanother embodiment, the length is 5-11 AA. In another embodiment, thelength is 6-11 AA. In another embodiment, the length is 7-11 AA. Inanother embodiment, the length is 8-11 AA. In another embodiment, thelength is 9-11 AA. In another embodiment, the length is 10-11 AA. Inanother embodiment, the length is 1-2 AA. In another embodiment, thelength is 1-3 AA. In another embodiment, the length is 1-4 AA. Inanother embodiment, the length is 1-5 AA. In another embodiment, thelength is 1-6 AA. In another embodiment, the length is 1-7 AA. Inanother embodiment, the length is 1-8 AA. In another embodiment, thelength is 1-9 AA. In another embodiment, the length is 1-10 AA. Inanother embodiment, the length is 2-3 AA. In another embodiment, thelength is 2-4 AA. In another embodiment, the length is 2-5 AA. Inanother embodiment, the length is 2-6 AA. In another embodiment, thelength is 2-7 AA. In another embodiment, the length is 2-8 AA. Inanother embodiment, the length is 2-9 AA. In another embodiment, thelength is 2-10 AA. In another embodiment, the length is 3-4 AA. Inanother embodiment, the length is 3-5 AA. In another embodiment, thelength is 3-6 AA. In another embodiment, the length is 3-7 AA. Inanother embodiment, the length is 3-8 AA. In another embodiment, thelength is 3-9 AA. In another embodiment, the length is 3-10 AA. Inanother embodiment, the length is 11-50 AA. In another embodiment, thelength is 12-50 AA. In another embodiment, the length is 11-15 AA. Inanother embodiment, the length is 11-20 AA. In another embodiment, thelength is 11-25 AA. In another embodiment, the length is 11-30 AA. Inanother embodiment, the length is 11-35 AA. In another embodiment, thelength is 11-40 AA. In another embodiment, the length is 11-60 AA. Inanother embodiment, the length is 11-70 AA. In another embodiment, thelength is 11-80 AA. In another embodiment, the length is 11-90 AA. Inanother embodiment, the length is 11-100 AA. In another embodiment, thelength is 11-150 AA. In another embodiment, the length is 15-20 AA. Inanother embodiment, the length is 15-25 AA. In another embodiment, thelength is 15-30 AA. In another embodiment, the length is 15-35 AA. Inanother embodiment, the length is 15-40 AA. In another embodiment, thelength is 15-60 AA. In another embodiment, the length is 15-70 AA. Inanother embodiment, the length is 15-80 AA. In another embodiment, thelength is 15-90 AA. In another embodiment, the length is 15-100 AA. Inanother embodiment, the length is 15-150 AA. In another embodiment, thelength is 20-25 AA. In another embodiment, the length is 20-30 AA. Inanother embodiment, the length is 20-35 AA. In another embodiment, thelength is 20-40 AA. In another embodiment, the length is 20-60 AA. Inanother embodiment, the length is 20-70 AA. In another embodiment, thelength is 20-80 AA. In another embodiment, the length is 20-90 AA. Inanother embodiment, the length is 20-100 AA. In another embodiment, thelength is 20-150 AA. In another embodiment, the length is 30-35 AA. Inanother embodiment, the length is 30-40 AA. In another embodiment, thelength is 30-60 AA. In another embodiment, the length is 30-70 AA. Inanother embodiment, the length is 30-80 AA. In another embodiment, thelength is 30-90 AA. In another embodiment, the length is 30-100 AA. Inanother embodiment, the length is 30-150 AA. Each possibility representsanother embodiment of the present invention.

In another embodiment, the length of the LLO fragment of methods andcompositions of the present invention is at least 484 AA. In anotherembodiment, the length is over 484 AA. In another embodiment, the lengthis at least 489 AA. In another embodiment, the length is over 489. Inanother embodiment, the length is at least 493 AA. In anotherembodiment, the length is over 493. In another embodiment, the length isat least 500 AA. In another embodiment, the length is over 500. Inanother embodiment, the length is at least 505 AA. In anotherembodiment, the length is over 505. In another embodiment, the length isat least 510 AA. In another embodiment, the length is over 510. Inanother embodiment, the length is at least 515 AA. In anotherembodiment, the length is over 515. In another embodiment, the length isat least 520 AA. In another embodiment, the length is over 520. Inanother embodiment, the length is at least 525 AA. In anotherembodiment, the length is over 520. When referring to the length of anLLO fragment herein, the signal sequence is included. Thus, thenumbering of the first cysteine in the CBD is 484, and the total numberof AA residues is 529. Each possibility represents another embodiment ofthe present invention.

In another embodiment, the present invention provides a recombinantprotein or polypeptide comprising (a) a mutated LLO protein, wherein themutated LLO protein contains an internal deletion, the internal deletioncomprising the cholesterol-binding domain of the mutated LLO protein;and (b) a heterologous peptide of interest. In another embodiment, thesequence of the cholesterol-binding domain is set forth in SEQ ID NO:18. In another embodiment, the internal deletion is an 11-50 amino acidinternal deletion. In another embodiment, the internal deletion isinactivating with regard to the hemolytic activity of the recombinantprotein or polypeptide. In another embodiment, the recombinant proteinor polypeptide exhibits a reduction in hemolytic activity relative towild-type LLO. Each possibility represents another embodiment of thepresent invention.

In another embodiment, the present invention provides a recombinantprotein or polypeptide comprising (a) a mutated LLO protein, wherein themutated LLO protein contains an internal deletion, the internal deletioncomprising comprises a residue selected from C484, W491, and W492 of themutated LLO protein; and (b) a heterologous peptide of interest. Inanother embodiment, the internal deletion is a 1-50 amino acid internaldeletion. In another embodiment, the sequence of the cholesterol-bindingdomain is set forth in SEQ ID NO: 18. In another embodiment, theinternal deletion is inactivating with regard to the hemolytic activityof the recombinant protein or polypeptide. In another embodiment, therecombinant protein or polypeptide exhibits a reduction in hemolyticactivity relative to wild-type LLO. Each possibility represents anotherembodiment of the present invention.

In another embodiment, the present invention provides a recombinantprotein or polypeptide comprising (a) a mutated LLO protein, wherein themutated LLO protein contains an internal deletion, the internal deletioncomprising a fragment of the cholesterol-binding domain of the mutatedLLO protein; and (b) a heterologous peptide of interest. In anotherembodiment, the internal deletion is a 1-11 amino acid internaldeletion. In another embodiment, the sequence of the cholesterol-bindingdomain is set forth in SEQ ID NO: 18. In another embodiment, theinternal deletion is inactivating with regard to the hemolytic activityof the recombinant protein or polypeptide. In another embodiment, therecombinant protein or polypeptide exhibits a reduction in hemolyticactivity relative to wild-type LLO. Each possibility represents anotherembodiment of the present invention.

In another embodiment, a peptide of the present invention is a fusionpeptide. In another embodiment, “fusion peptide” refers to a peptide orpolypeptide comprising two or more proteins linked together by peptidebonds or other chemical bonds. In another embodiment, the proteins arelinked together directly by a peptide or other chemical bond. In anotherembodiment, the proteins are linked together with one or more AA (e.g. a“spacer”) between the two or more proteins. Each possibility representsa separate embodiment of the present invention.

As provided herein, a mutant LLO protein was created wherein residuesC484, W491, and W492 of LLO were substituted with a CTL epitope from theantigen NY-ESO-1 (Example 6). The mutated LLO protein, mutLLO, could beexpressed and purified in an E. coli expression system (Example 7) andexhibited substantially reduced hemolytic activity relative to wild-typeLLO (Example 8).

The length of the internal deletion of methods and compositions of thepresent invention is, in another embodiment, 1-50 AA. In anotherembodiment, the length is 1-11 AA. In another embodiment, the length is2-11 AA. In another embodiment, the length is 3-11 AA. In anotherembodiment, the length is 4-11 AA. In another embodiment, the length is5-11 AA. In another embodiment, the length is 6-11 AA. In anotherembodiment, the length is 7-11 AA. In another embodiment, the length is8-11 AA. In another embodiment, the length is 9-11 AA. In anotherembodiment, the length is 10-11 AA. In another embodiment, the length is1-2 AA. In another embodiment, the length is 1-3 AA. In anotherembodiment, the length is 1-4 AA. In another embodiment, the length is1-5 AA. In another embodiment, the length is 1-6 AA. In anotherembodiment, the length is 1-7 AA. In another embodiment, the length is1-8 AA. In another embodiment, the length is 1-9 AA. In anotherembodiment, the length is 1-10 AA. In another embodiment, the length is2-3 AA. In another embodiment, the length is 2-4 AA. In anotherembodiment, the length is 2-5 AA. In another embodiment, the length is2-6 AA. In another embodiment, the length is 2-7 AA. In anotherembodiment, the length is 2-8 AA. In another embodiment, the length is2-9 AA. In another embodiment, the length is 2-10 AA. In anotherembodiment, the length is 3-4 AA. In another embodiment, the length is3-5 AA. In another embodiment, the length is 3-6 AA. In anotherembodiment, the length is 3-7 AA. In another embodiment, the length is3-8 AA. In another embodiment, the length is 3-9 AA. In anotherembodiment, the length is 3-10 AA. In another embodiment, the length is11-50 AA. In another embodiment, the length is 12-50 AA. In anotherembodiment, the length is 11-15 AA. In another embodiment, the length is11-20 AA. In another embodiment, the length is 11-25 AA. In anotherembodiment, the length is 11-30 AA. In another embodiment, the length is11-35 AA. In another embodiment, the length is 11-40 AA. In anotherembodiment, the length is 11-60 AA. In another embodiment, the length is11-70 AA. In another embodiment, the length is 11-80 AA. In anotherembodiment, the length is 11-90 AA. In another embodiment, the length is11-100 AA. In another embodiment, the length is 11-150 AA. In anotherembodiment, the length is 15-20 AA. In another embodiment, the length is15-25 AA. In another embodiment, the length is 15-30 AA. In anotherembodiment, the length is 15-35 AA. In another embodiment, the length is15-40 AA. In another embodiment, the length is 15-60 AA. In anotherembodiment, the length is 15-70 AA. In another embodiment, the length is15-80 AA. In another embodiment, the length is 15-90 AA. In anotherembodiment, the length is 15-100 AA. In another embodiment, the lengthis 15-150 AA. In another embodiment, the length is 20-25 AA. In anotherembodiment, the length is 20-30 AA. In another embodiment, the length is20-35 AA. In another embodiment, the length is 20-40 AA. In anotherembodiment, the length is 20-60 AA. In another embodiment, the length is20-70 AA. In another embodiment, the length is 20-80 AA. In anotherembodiment, the length is 20-90 AA. In another embodiment, the length is20-100 AA. In another embodiment, the length is 20-150 AA. In anotherembodiment, the length is 30-35 AA. In another embodiment, the length is30-40 AA. In another embodiment, the length is 30-60 AA. In anotherembodiment, the length is 30-70 AA. In another embodiment, the length is30-80 AA. In another embodiment, the length is 30-90 AA. In anotherembodiment, the length is 30-100 AA. In another embodiment, the lengthis 30-150 AA. Each possibility represents another embodiment of thepresent invention.

In another embodiment, the mutated LLO protein of the present inventionthat comprises an internal deletion is full length except for theinternal deletion. In another embodiment, the mutated LLO proteincomprises an additional internal deletion. In another embodiment, themutated LLO protein comprises more than one additional internaldeletion. In another embodiment, the mutated LLO protein is truncatedfrom the C-terminal end. In another embodiment, the mutated LLO proteinis truncated from the N-terminal end. Each possibility representsanother embodiment of the present invention.

The internal deletion of methods and compositions of the presentinvention comprises, in another embodiment, residue C484 of SEQ ID NO:37. In another embodiment, the internal deletion comprises acorresponding cysteine residue of a homologous LLO protein. In anotherembodiment, the internal deletion comprises residue W491 of SEQ ID NO:37. In another embodiment, the internal deletion comprises acorresponding tryptophan residue of a homologous LLO protein. In anotherembodiment, the internal deletion comprises residue W492 of SEQ ID NO:37. In another embodiment, the internal deletion comprises acorresponding tryptophan residue of a homologous LLO protein. Methodsfor identifying corresponding residues of a homologous protein are wellknown in the art, and include, for example, sequence alignment. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the internal deletion comprises residues C484 andW491. In another embodiment, the internal deletion comprises residuesC484 and W492. In another embodiment, the internal deletion comprisesresidues W491 and W492. In another embodiment, the internal deletioncomprises residues C484, W491, and W492. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the internal deletion of methods and compositionsof the present invention comprises the CBD of the mutated LLO protein orfragment thereof. For example, an internal deletion consisting ofresidues 470-500, 470-510, or 480-500 of SEQ ID NO: 37 comprises the CBDthereof (residues 483-493). In another embodiment, the internal deletionis a fragment of the CBD of the mutated LLO protein or fragment thereof.For example, residues 484-492, 485-490, and 486-488 are all fragments ofthe CBD of SEQ ID NO: 37. In another embodiment, the internal deletionoverlaps the CBD of the mutated LLO protein or fragment thereof. Forexample, an internal deletion consisting of residues 470-490, 480-488,490-500, or 486-510 of SEQ ID NO: 37 comprises the CBD thereof. Eachpossibility represents a separate embodiment of the present invention.

“Hemolytic” refers, in another embodiment, to ability to lyse aeukaryotic cell. In another embodiment, the eukaryotic cell is a redblood cell. In another embodiment, the eukaryotic cell is any other typeof eukaryotic cell known in the art. In another embodiment, hemolyticactivity is measured at an acidic pH. In another embodiment, hemolyticactivity is measured at physiologic pH. In another embodiment, hemolyticactivity is measured at pH 5.5. In another embodiment, hemolyticactivity is measured at pH 7.4. In another embodiment, hemolyticactivity is measured at any other pH known in the art.

In another embodiment, a recombinant protein or polypeptide of methodsand compositions of the present invention exhibits a greater than100-fold reduction in hemolytic activity relative to wild-type LLO. Inanother embodiment, the recombinant protein or polypeptide exhibits agreater than 50-fold reduction in hemolytic activity. In anotherembodiment, the reduction is greater than 30-fold. In anotherembodiment, the reduction is greater than 40-fold. In anotherembodiment, the reduction is greater than 60-fold. In anotherembodiment, the reduction is greater than 70-fold. In anotherembodiment, the reduction is greater than 80-fold. In anotherembodiment, the reduction is greater than 90-fold. In anotherembodiment, the reduction is greater than 120-fold. In anotherembodiment, the reduction is greater than 150-fold. In anotherembodiment, the reduction is greater than 200-fold. In anotherembodiment, the reduction is greater than 250-fold. In anotherembodiment, the reduction is greater than 300-fold. In anotherembodiment, the reduction is greater than 400-fold. In anotherembodiment, the reduction is greater than 500-fold. In anotherembodiment, the reduction is greater than 600-fold. In anotherembodiment, the reduction is greater than 800-fold. In anotherembodiment, the reduction is greater than 1000-fold. In anotherembodiment, the reduction is greater than 1200-fold. In anotherembodiment, the reduction is greater than 1500-fold. In anotherembodiment, the reduction is greater than 2000-fold. In anotherembodiment, the reduction is greater than 3000-fold. In anotherembodiment, the reduction is greater than 5000-fold.

In another embodiment, the reduction is at least 100-fold. In anotherembodiment, the reduction is at least 50-fold. In another embodiment,the reduction is at least 30-fold. In another embodiment, the reductionis at least 40-fold. In another embodiment, the reduction is at least60-fold. In another embodiment, the reduction is at least 70-fold. Inanother embodiment, the reduction is at least 80-fold. In anotherembodiment, the reduction is at least 90-fold. In another embodiment,the reduction is at least 120-fold. In another embodiment, the reductionis at least 150-fold. In another embodiment, the reduction is at least200-fold. In another embodiment, the reduction is at least 250-fold. Inanother embodiment, the reduction is at least 300-fold. In anotherembodiment, the reduction is at least 400-fold. In another embodiment,the reduction is at least 500-fold. In another embodiment, the reductionis at least 600-fold. In another embodiment, the reduction is at least800-fold. In another embodiment, the reduction is at least 1000-fold. Inanother embodiment, the reduction is at least 1200-fold. In anotherembodiment, the reduction is at least 1500-fold. In another embodiment,the reduction is at least 2000-fold. In another embodiment, thereduction is at least 3000-fold. In another embodiment, the reduction isat least 5000-fold.

Methods of determining hemolytic activity are well known in the art, andare described, for example, in the Examples herein, and in Portnoy D Aet al, (J Exp Med Vol 167:1459-1471, 1988) and Dancz C E et al (JBacteriol. 184: 5935-5945, 2002).

“Inactivating mutation” with respect to hemolytic activity refers, inanother embodiment, to a mutation that abolishes detectable hemolyticactivity. In another embodiment, the term refers to a mutation thatabolishes hemolytic activity at pH 5.5. In another embodiment, the termrefers to a mutation that abolishes hemolytic activity at pH 7.4. Inanother embodiment, the term refers to a mutation that significantlyreduces hemolytic activity at pH 5.5. In another embodiment, the termrefers to a mutation that significantly reduces hemolytic activity at pH7.4. In another embodiment, the term refers to a mutation thatsignificantly reduces hemolytic activity at pH 5.5. In anotherembodiment, the term refers to any other type of inactivating mutationwith respect to hemolytic activity. Each possibility represents anotherembodiment of the present invention.

In another embodiment, the sequence of the cholesterol-binding domain ofmethods and compositions of the present invention is set forth in SEQ IDNO: 18. In another embodiment, the CBD is any other LLO CBD known in theart. Each possibility represents another embodiment of the presentinvention.

The non-LLO sequence of methods and compositions of the presentinvention is, in another embodiment, a heterologous sequence. In anotherembodiment, the non-LLO sequence is a synthetic sequence. In anotherembodiment, the non-LLO sequence is a non-naturally occurring sequence.In another embodiment, the non-LLO sequence is a non-Listeria sequence.In another embodiment, the non-LLO sequence is a non-Listeriamonocytogenes sequence. In one embodiment, the compositions of thepresent invention comprise a mutated LLO in which there is asubstitution of an amino acid peptide comprising a CBD for an amino acidcomprising a non-LLO peptide and further comprising a heterologousantigen fused to said mutated LLO. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the mutated LLO protein or fragment thereof ofmethods and compositions of the present invention comprises the signalpeptide thereof. In another embodiment, the mutated LLO protein orfragment thereof comprises a signal peptide of a wild-type LLO protein.In another embodiment, the signal peptide is a short (3-60 amino acidlong) peptide chain that directs the post-translational transport of aprotein. In another embodiment, signal peptides are also targetingsignals, signal sequences, transit peptides, or localization signals. Inanother embodiment, the amino acid sequences of signal peptides directproteins to certain organelles such as the nucleus, mitochondrialmatrix, endoplasmic reticulum, chloroplast, apoplast or peroxisome. Inanother embodiment, the mutated LLO protein contains a signal sequenceof a wild-type LLO protein. In another embodiment, the mutated LLOprotein lacks a signal peptide. In another embodiment, the mutated LLOprotein lacks a signal sequence. In another embodiment, the signalpeptide is unaltered with respect to the wild-type LLO protein fromwhich the mutated LLO protein or fragment thereof was derived. Inanother embodiment, the signal peptide is on N-terminal end ofrecombinant protein or polypeptide. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the mutated LLO protein or fragment thereof ofmethods and compositions of the present invention comprises a PEST-likepeptide sequence. In another embodiment, the PEST-like peptide sequenceis an LLO PEST-like peptide sequence. In another embodiment, the aminoacid sequence of the PEST-like peptide sequence is forth in SEQ ID NO:63. Each possibility represents a separate embodiment of the presentinvention.

In one embodiment, PEST sequences are sequences that are rich inprolines (P), glutamic acids (E), serines (S) and threonines (T),generally, but not always, flanked by clusters containing severalpositively charged amino acids, have rapid intracellular half-lives(Rogers et al., 1986, Science 234:364-369). In another embodiment, PESTsequences target the protein to the ubiquitin-proteosome pathway fordegradation (Rechsteiner and Rogers TIBS 1996 21:267-271), which in oneembodiment, is a pathway also used by eukaryotic cells to generateimmunogenic peptides that bind to MHC class I. PEST sequences areabundant among eukaryotic proteins that give rise to immunogenicpeptides (Realini et al. FEBS Lett. 1994 348:109-113). Although PESTsequences are usually found in eurkaryotic proteins, a PEST-likesequence rich in the amino acids proline (P), glutamic acid (E), serine(S) and threonine (T) was identified at the amino terminus of theprokaryotic Listeria LLO protein and demonstrated to be essential for L.monocytogenes pathogenicity (Decatur, A. L. and Portnoy, D. A. Science2000 290:992-995). In one embodiment, the presence of this PEST-likesequence in LLO targets the protein for destruction by proteolyticmachinery of the host cell so that once the LLO has served its functionand facilitated the escape of L. monocytogenes from the phagolysosomalvacuole, it is destroyed before it damages the cells.

In another embodiment, the immune response to an antigen can be enhancedby fusion of the antigen to a non-hemolytic truncated form oflisteriolysin O (ΔLLO). In one embodiment, the observed enhanced cellmediated immunity and anti-tumor immunity of the fusion protein resultsfrom the PEST-like sequence present in LLO which targets the antigen forprocessing.

In another embodiment, the non-LLO peptide that replaces the mutatedregion of the recombinant protein or polypeptide comprises an antigenicpeptide of interest. In another embodiment, the antigenic peptide is acytotoxic T lymphocyte (CTL) epitope. In another embodiment, theantigenic peptide is a CD4⁺ T cell epitope. In another embodiment, theantigenic peptide is any other type of peptide known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a vaccinecomprising an adjuvant and a recombinant protein or polypeptide of thepresent invention, wherein an antigenic peptide of interest replaces themutated region. In another embodiment, the present invention provides animmunogenic composition comprising the recombinant protein orpolypeptide. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a nucleotidemolecule encoding a recombinant protein or polypeptide of the presentinvention, wherein an antigenic peptide of interest replaces the mutatedregion.

In another embodiment, a recombinant protein or polypeptide of methodsand compositions of the present invention further comprises aheterologous peptide of interest. In another embodiment, theheterologous peptide of interest is fused to the mutated LLO or fragmentthereof. In another embodiment, the heterologous peptide of interest isfused to the C-terminal end of the mutated LLO or fragment thereof. Inanother embodiment, the heterologous peptide of interest is embeddedwithin the mutated LLO or fragment thereof, e.g. at a location otherthan the mutated region comprising or overlapping the CBD. In anotherembodiment, the heterologous peptide of interest is inserted into thesequence of the mutated LLO or fragment thereof, e.g. at a locationother than the mutated region comprising or overlapping the CBD. Inanother embodiment, the heterologous peptide of interest is substitutedfor sequence of the mutated LLO or fragment thereof, e.g. at a locationother than the mutated region comprising or overlapping the CBD. Thus,in one embodiment, the recombinant protein or polypeptide of the presentinvention comprises a mutated LLO or fragment thereof fused orconjugated to an antigenic peptide or protein.

In another embodiment, the present invention provides a vaccinecomprising an adjuvant and a recombinant protein or polypeptide of thepresent invention, wherein the recombinant protein or polypeptidefurther comprises a heterologous peptide of interest. In anotherembodiment, the present invention provides an immunogenic compositioncomprising the recombinant protein or polypeptide. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a nucleotidemolecule encoding a recombinant protein or polypeptide of the presentinvention, wherein the recombinant protein or polypeptide furthercomprises a heterologous peptide of interest.

In another embodiment, the LLO protein or fragment thereof of methodsand compositions of the present invention is on the N-terminal end of arecombinant protein or polypeptide of the present invention. In anotherembodiment, the LLO protein or fragment thereof is in any other positionin the recombinant protein or polypeptide. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the present invention provides a compositioncomprising a recombinant protein or polypeptide of the present inventionand a heterologous peptide of interest. In another embodiment, thepresent invention provides a composition comprising a recombinantprotein or polypeptide of the present invention and a heterologousantigenic peptide of interest. In another embodiment, the recombinantprotein or polypeptide is not covalently bound to the heterologouspeptide of interest. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the present invention provides a vaccinecomprising an adjuvant, a recombinant protein or polypeptide of thepresent invention, and a heterologous peptide of interest. In anotherembodiment, the present invention provides a composition comprising anadjuvant, a recombinant protein or polypeptide of the present invention,and a heterologous antigenic peptide of interest. In another embodiment,the recombinant protein or polypeptide is not covalently bound to theheterologous peptide of interest. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a vaccinecomprising an adjuvant and a recombinant protein or polypeptide of thepresent invention. In another embodiment, the present invention providesan immunogenic composition comprising the recombinant protein orpolypeptide. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a nucleotidemolecule encoding a recombinant protein or polypeptide of the presentinvention.

In another embodiment, the present invention provides a vaccinecomprising a nucleotide molecule of the present invention and anadjuvant.

In another embodiment, the present invention provides a recombinantvaccine vector comprising a nucleotide molecule of the presentinvention.

In another embodiment, the present invention provides a recombinantvaccine vector encoding a recombinant protein or polypeptide of thepresent invention.

In another embodiment, the present invention provides a recombinantListeria strain comprising a recombinant protein or polypeptide of thepresent invention. In another embodiment, the present invention providesa recombinant Listeria strain expressing a recombinant protein orpolypeptide of the present invention. In another embodiment, the presentinvention provides a recombinant Listeria strain encoding a recombinantprotein or polypeptide of the present invention. In another embodiment,the present invention provides a recombinant Listeria strain comprisinga recombinant nucleotide encoding a recombinant polypeptide of thepresent invention. In another embodiment, the Listeria vaccine strain isthe species Listeria monocytogenes (LM). Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the present invention provides a cell comprisinga vector of the present invention. Methods for producing cellscomprising vectors and/or exogenous nucleic acids are well-known in theart. See, for example, Sambrook et al. (1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inAusubel et al. (1997, Current Protocols in Molecular Biology, John Wiley& Sons, New York).

In another embodiment, the present invention provides a vaccinecomprising a nucleotide molecule of the present invention. In anotherembodiment, the present invention provides an immunogenic compositioncomprising the nucleotide molecule. Each possibility represents aseparate embodiment of the present invention.

The adjuvant utilized in methods and compositions of the presentinvention is, in another embodiment, a granulocyte/macrophagecolony-stimulating factor (GM-CSF) protein. In another embodiment, theadjuvant comprises a GM-CSF protein. In another embodiment, the adjuvantis a nucleotide molecule encoding GM-CSF. In another embodiment, theadjuvant comprises a nucleotide molecule encoding GM-CSF. In anotherembodiment, the adjuvant is saponin QS21. In another embodiment, theadjuvant comprises saponin QS21. In another embodiment, the adjuvant ismonophosphoryl lipid A. In another embodiment, the adjuvant comprisesmonophosphoryl lipid A. In another embodiment, the adjuvant is SBAS2. Inanother embodiment, the adjuvant comprises SBAS2. In another embodiment,the adjuvant is an unmethylated CpG-containing oligonucleotide. Inanother embodiment, the adjuvant comprises an unmethylatedCpG-containing oligonucleotide. In another embodiment, the adjuvant isan immune-stimulating cytokine. In another embodiment, the adjuvantcomprises an immune-stimulating cytokine. In another embodiment, theadjuvant is a nucleotide molecule encoding an immune-stimulatingcytokine. In another embodiment, the adjuvant comprises a nucleotidemolecule encoding an immune-stimulating cytokine. In another embodiment,the adjuvant is or comprises a quill glycoside. In another embodiment,the adjuvant is or comprises a bacterial mitogen. In another embodiment,the adjuvant is or comprises a bacterial toxin. In another embodiment,the adjuvant is or comprises any other adjuvant known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forinducing an immune response in a subject, comprising administering tothe subject a recombinant protein or polypeptide of the presentinvention, wherein the recombinant protein or polypeptide contains anantigenic peptide of interest, thereby inducing an immune responseagainst an antigenic peptide of interest.

In another embodiment, the present invention provides a method forinducing an immune response in a subject, comprising administering tothe subject a recombinant protein or polypeptide of the presentinvention, wherein the recombinant protein or polypeptide contains aheterologous peptide of interest, thereby inducing an immune responseagainst a heterologous peptide of interest.

In another embodiment, the present invention provides a method forinducing an immune response in a subject, comprising administering tothe subject a recombinant vaccine vector of the present invention,wherein the recombinant vaccine vector comprises or encodes arecombinant protein or polypeptide that comprises a heterologousantigenic peptide of interest, thereby inducing an immune responseagainst the antigenic peptide of interest.

In another embodiment, the present invention provides a method forinducing an immune response in a subject, comprising administering tothe subject a recombinant vaccine vector of the present invention,wherein the recombinant vaccine vector comprises or encodes arecombinant protein or polypeptide that comprises a heterologous peptideof interest, thereby inducing an immune response against theheterologous peptide of interest.

In another embodiment, the present invention provides a method forinducing an immune response in a subject, comprising administering tothe subject a recombinant Listeria strain of the present invention,wherein the recombinant Listeria strain comprises or encodes arecombinant protein or polypeptide that comprises a heterologous peptideof interest, thereby inducing an immune response against theheterologous peptide of interest.

In another embodiment, the present invention provides a method forinducing an immune response in a subject, comprising administering tothe subject a recombinant Listeria strain of the present invention,wherein the recombinant Listeria strain comprises or encodes arecombinant protein or polypeptide that comprises a heterologousantigenic peptide of interest, thereby inducing an immune responseagainst the heterologous peptide of interest.

In another embodiment of methods and compositions of the presentinvention, a peptide or nucleotide molecule of the present invention isadministered to a subject having a lymphoma, cancer cell, or infectiousdisease expressing a target antigen of the present invention. In anotherembodiment, the peptide or nucleotide molecule is administered ex vivoto cells of a subject. In another embodiment, the peptide isadministered to a lymphocyte donor; lymphocytes from the donor are thenadministered, in another embodiment, to a subject. In anotherembodiment, the peptide is administered to an antibody or lymphocytedonor; antiserum or lymphocytes from the donor is then administered, inanother embodiment, to a subject. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the heterologous peptide of interest is afull-length protein, which in one embodiment, comprises an antigenicpeptide. In one embodiment, the protein is an NY-ESO-1 protein. Inanother embodiment, the protein is a Human Papilloma Virus (HPV) E7protein. In another embodiment, the protein is a B-cell receptor (BCR)protein. In another embodiment, the heterologous peptide of interest isan antigenic peptide.

The antigenic peptide of interest of methods and compositions of thepresent invention is, in another embodiment, an NY-ESO-1 peptide.

In another embodiment, the present invention provides a recombinantnucleotide molecule encoding an NY-ESO-1-containing peptide of thepresent invention.

In another embodiment, the present invention provides a compositioncomprising a mutant-LLO containing recombinant protein or polypeptide ofthe present invention and an NY-ESO-1 peptide.

In another embodiment, the present invention provides a recombinantvaccine vector encoding an NY-ESO-1-containing peptide of the presentinvention.

In another embodiment, the present invention provides a recombinantListeria strain encoding an NY-ESO-1-containing peptide of the presentinvention.

In one embodiment, NY-ESO-1 is a “cancer-testis” antigen expressed inepithelial ovarian cancer (EOC). In another embodiment, NY-ESO-1 isexpressed in metastatic melanoma, breast cancer, lung cancer, esophagealcancer, which in one embodiment, is esophageal squamous cell carcinoma,or a combination thereof. Therefore, in one embodiment, the compositionsand methods of the present invention comprising NY-ESO-1 areparticularly useful in the prevention or treatment of theabove-mentioned cancers.

In another embodiment, the present invention provides a method ofproducing a recombinant protein or polypeptide of the present inventioncomprising the step of chemically conjugating a peptide comprising saidmutated LLO protein or mutated N-terminal LLO fragment to a peptidecomprising said heterologous peptide of interest. In another embodiment,the present invention provides a method of producing a recombinantprotein or polypeptide of the present invention comprising the step oftranslating said recombinant protein or polypeptide from a nucleotidemolecule encoding same. In another embodiment, the present inventionprovides a product made by one or more of the processes describedherein.

In another embodiment, the present invention provides a method forinducing an immune response in a subject against an NY-ESO-1-expressingcancer cell, the method comprising the step of administering to thesubject an NY-ESO-1 antigen-containing recombinant peptide, protein orpolypeptide of the present invention, thereby inducing an immuneresponse against an NY-ESO-1-expressing cancer cell. In anotherembodiment, the cancer cell is an ovarian melanoma cell. In anotherembodiment, the cancer cell is a lung cancer cell. In anotherembodiment, the cancer cell is any other NY-ESO-1-expressing cancer cellknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the present invention provides a method fortreating an NY-ESO-1-expressing tumor in a subject, the methodcomprising the step of administering to the subject an NY-ESO-1antigen-containing recombinant peptide, protein or polypeptide of thepresent invention, thereby treating an NY-ESO-1-expressing tumor in asubject. In another embodiment, the tumor is an ovarian melanoma tumor.In another embodiment, the tumor is a lung cancer tumor. In anotherembodiment, the tumor is any other NY-ESO-1-expressing tumor known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method forinhibiting an NY-ESO-1-expressing tumor in a subject, the methodcomprising the step of administering to the subject an NY-ESO-1antigen-containing recombinant peptide, protein or polypeptide of thepresent invention, thereby inhibiting an NY-ESO-1-expressing tumor in asubject. In another embodiment, the tumor is an ovarian melanoma tumor.In another embodiment, the tumor is a lung cancer tumor. In anotherembodiment, the tumor is any other NY-ESO-1-expressing tumor known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method forsuppressing an NY-ESO-1-expressing tumor in a subject, the methodcomprising the step of administering to the subject an NY-ESO-1antigen-containing recombinant peptide, protein or polypeptide of thepresent invention, thereby suppressing an NY-ESO-1-expressing tumor in asubject. In another embodiment, the tumor is an ovarian melanoma tumor.In another embodiment, the tumor is a lung cancer tumor. In anotherembodiment, the tumor is any other NY-ESO-1-expressing tumor known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method forinducing regression of an NY-ESO-1-expressing tumor in a subject, themethod comprising the step of administering to the subject an NY-ESO-1antigen-containing recombinant peptide, protein or polypeptide of thepresent invention, thereby inducing regression of an NY-ESO-1-expressingtumor in a subject. In another embodiment, the tumor is an ovarianmelanoma tumor. In another embodiment, the tumor is a lung cancer tumor.In another embodiment, the tumor is any other NY-ESO-1-expressing tumorknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the present invention provides a method forreducing an incidence of an NY-ESO-1-expressing tumor in a subject, themethod comprising the step of administering to the subject an NY-ESO-1antigen-containing recombinant peptide, protein or polypeptide of thepresent invention, thereby reducing an incidence of anNY-ESO-1-expressing tumor in a subject. In another embodiment, the tumoris an ovarian melanoma tumor. In another embodiment, the tumor is a lungcancer tumor. In another embodiment, the tumor is any otherNY-ESO-1-expressing tumor known in the art. Each possibility representsa separate embodiment of the present invention.

In another embodiment, the present invention provides a method forprotecting a subject against an NY-ESO-1-expressing tumor, the methodcomprising the step of administering to the subject an NY-ESO-1antigen-containing recombinant peptide, protein or polypeptide of thepresent invention, thereby protecting a subject against anNY-ESO-1-expressing tumor. In another embodiment, the tumor is anovarian melanoma tumor. In another embodiment, the tumor is a lungcancer tumor. In another embodiment, the tumor is any otherNY-ESO-1-expressing tumor known in the art. Each possibility representsa separate embodiment of the present invention.

In another embodiment, the present invention provides a method forinducing an immune response in a subject against an NY-ESO-1-expressingcancer cell, the method comprising the step of administering to thesubject an NY-ESO-1 antigen-expressing vaccine vector of the presentinvention, thereby inducing an immune response against anNY-ESO-1-expressing cancer cell. In another embodiment, the cancer cellis an ovarian melanoma cell. In another embodiment, the cancer cell is alung cancer cell. In another embodiment, the cancer cell is any otherNY-ESO-1-expressing cancer cell known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method fortreating or reducing an incidence of an NY-ESO-1-expressing tumor in asubject, the method comprising the step of administering to the subjectan NY-ESO-1 antigen-expressing vaccine vector of the present invention,thereby treating or reducing an incidence of an NY-ESO-1-expressingtumor in a subject. In another embodiment, the tumor is an ovarianmelanoma tumor. In another embodiment, the tumor is a lung cancer tumor.In another embodiment, the tumor is any other NY-ESO-1-expressing tumorknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the present invention provides a method forinducing regression of an NY-ESO-1-expressing tumor in a subject, themethod comprising the step of administering to the subject an NY-ESO-1antigen-expressing vaccine vector of the present invention, therebyinducing regression of an NY-ESO-1-expressing tumor in a subject. Inanother embodiment, the tumor is an ovarian melanoma tumor. In anotherembodiment, the tumor is a lung cancer tumor. In another embodiment, thetumor is any other NY-ESO-1-expressing tumor known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forprotecting a subject against an NY-ESO-1-expressing tumor, the methodcomprising the step of administering to the subject an NY-ESO-1antigen-expressing vaccine vector of the present invention, therebyprotecting a subject against an NY-ESO-1-expressing tumor. In anotherembodiment, the tumor is an ovarian melanoma tumor. In anotherembodiment, the tumor is a lung cancer tumor. In another embodiment, thetumor is any other NY-ESO-1-expressing tumor known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forinducing an immune response in a subject against an NY-ESO-1-expressingcancer cell, the method comprising the step of administering to thesubject an NY-ESO-1 antigen-encoding nucleotide molecule of the presentinvention, thereby inducing an immune response against anNY-ESO-1-expressing cancer cell. In another embodiment, the cancer cellis an ovarian melanoma cell. In another embodiment, the cancer cell is alung cancer cell. In another embodiment, the cancer cell is any otherNY-ESO-1-expressing cancer cell known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method fortreating or reducing an incidence of an NY-ESO-1-expressing tumor in asubject, the method comprising the step of administering to the subjectan NY-ESO-1 antigen-encoding nucleotide molecule of the presentinvention, thereby treating or reducing an incidence of anNY-ESO-1-expressing tumor in a subject. In another embodiment, the tumoris an ovarian melanoma tumor. In another embodiment, the tumor is a lungcancer tumor. In another embodiment, the tumor is any otherNY-ESO-1-expressing tumor known in the art. Each possibility representsa separate embodiment of the present invention.

In another embodiment, the present invention provides a method forinhibiting an NY-ESO-1-expressing tumor in a subject, the methodcomprising the step of administering to the subject an NY-ESO-1antigen-encoding nucleotide molecule of the present invention, therebyinhibiting an NY-ESO-1-expressing tumor in a subject. In anotherembodiment, the tumor is an ovarian melanoma tumor. In anotherembodiment, the tumor is a lung cancer tumor. In another embodiment, thetumor is any other NY-ESO-1-expressing tumor known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forsuppressing an NY-ESO-1-expressing tumor in a subject, the methodcomprising the step of administering to the subject an NY-ESO-1antigen-encoding nucleotide molecule of the present invention, therebyinhibiting an NY-ESO-1-expressing tumor in a subject. In anotherembodiment, the tumor is an ovarian melanoma tumor. In anotherembodiment, the tumor is a lung cancer tumor. In another embodiment, thetumor is any other NY-ESO-1-expressing tumor known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forinducing regression of an NY-ESO-1-expressing tumor in a subject, themethod comprising the step of administering to the subject an NY-ESO-1antigen-encoding nucleotide molecule of the present invention, therebyinducing regression of an NY-ESO-1-expressing tumor in a subject. Inanother embodiment, the tumor is an ovarian melanoma tumor. In anotherembodiment, the tumor is a lung cancer tumor. In another embodiment, thetumor is any other NY-ESO-1-expressing tumor known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forprotecting a subject against an NY-ESO-1-expressing tumor, the methodcomprising the step of administering to the subject an NY-ESO-1antigen-encoding nucleotide molecule of the present invention, therebyprotecting a subject against an NY-ESO-1-expressing tumor. In anotherembodiment, the tumor is an ovarian melanoma tumor. In anotherembodiment, the tumor is a lung cancer tumor. In another embodiment, thetumor is any other NY-ESO-1-expressing tumor known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forinducing an immune response in a subject against an NY-ESO-1-expressingcancer cell, the method comprising the step of administering to thesubject an NY-ESO-1 antigen-expressing recombinant Listeria strain ofthe present invention, thereby inducing an immune response against anNY-ESO-1-expressing cancer cell. In another embodiment, the cancer cellis an ovarian melanoma cell. In another embodiment, the cancer cell is alung cancer cell. In another embodiment, the cancer cell is any otherNY-ESO-1-expressing cancer cell known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method fortreating or reducing an incidence of an NY-ESO-1-expressing tumor in asubject, the method comprising the step of administering to the subjectan NY-ESO-1 antigen-expressing recombinant Listeria strain of thepresent invention, thereby treating or reducing an incidence of anNY-ESO-1-expressing tumor in a subject. In another embodiment, the tumoris an ovarian melanoma tumor. In another embodiment, the tumor is a lungcancer tumor. In another embodiment, the tumor is any otherNY-ESO-1-expressing tumor known in the art. Each possibility representsa separate embodiment of the present invention.

In another embodiment, the present invention provides a method forinhibiting an NY-ESO-1-expressing tumor in a subject, the methodcomprising the step of administering to the subject an NY-ESO-1antigen-expressing recombinant Listeria strain of the present invention,thereby treating or reducing an incidence of an NY-ESO-1-expressingtumor in a subject. In another embodiment, the tumor is an ovarianmelanoma tumor. In another embodiment, the tumor is a lung cancer tumor.In another embodiment, the tumor is any other NY-ESO-1-expressing tumorknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the present invention provides a method forsuppressing an NY-ESO-1-expressing tumor in a subject, the methodcomprising the step of administering to the subject an NY-ESO-1antigen-expressing recombinant Listeria strain of the present invention,thereby treating or reducing an incidence of an NY-ESO-1-expressingtumor in a subject. In another embodiment, the tumor is an ovarianmelanoma tumor. In another embodiment, the tumor is a lung cancer tumor.In another embodiment, the tumor is any other NY-ESO-1-expressing tumorknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the present invention provides a method forinducing regression of an NY-ESO-1-expressing tumor in a subject, themethod comprising the step of administering to the subject an NY-ESO-1antigen-expressing recombinant Listeria strain of the present invention,thereby inducing regression of an NY-ESO-1-expressing tumor in asubject. In another embodiment, the tumor is an ovarian melanoma tumor.In another embodiment, the tumor is a lung cancer tumor. In anotherembodiment, the tumor is any other NY-ESO-1-expressing tumor known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method forprotecting a subject against an NY-ESO-1-expressing tumor, the methodcomprising the step of administering to the subject an NY-ESO-1antigen-expressing recombinant Listeria strain of the present invention,thereby protecting a subject against an NY-ESO-1-expressing tumor. Inanother embodiment, the tumor is an ovarian melanoma tumor. In anotherembodiment, the tumor is a lung cancer tumor. In another embodiment, thetumor is any other NY-ESO-1-expressing tumor known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forinducing an immune response against an NY-ESO-1 epitope, comprising thestep of administering to the subject an NY-ESO-1 antigen-containingrecombinant peptide, protein or polypeptide of the present invention,thereby inducing an immune response against an NY-ESO-1 epitope.

In another embodiment, the present invention provides a method forinducing an immune response against an NY-ESO-1 antigen, comprising thestep of administering to the subject a recombinant peptide, protein orpolypeptide of the present invention containing said NY-ESO-1 antigen,thereby inducing an immune response against an NY-ESO-1 epitope.

In one embodiment, a NY-ESO-1 epitope for use in the compositions andmethods of the present invention is ASGPGGGAPR: 53-62 (A31), ARGPESRLL:80-88 (Cw6), LAMPFATPM: 92-100 (Cw3), MPFATPMEA: 94-102 (B35, B51),TVSGNILTR: 127-136 (A68), TVSGNILT: 127-135 (Cw15), SLLMWITQC: 157-165(A2; Example 6), or another NY-ESO-1 epitope known in the art.

In another embodiment, the present invention provides a method forinducing an immune response against an NY-ESO-1-expressing target cell,comprising the step of administering to the subject an NY-ESO-1antigen-containing recombinant peptide, protein or polypeptide of thepresent invention, thereby inducing an immune response against anNY-ESO-1-expressing target cell. In another embodiment, the target cellis an ovarian melanoma cell. In another embodiment, the target cell is alung cancer cell. In another embodiment, the target cell is any otherNY-ESO-1-expressing cell known in the art. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the target NY-ESO-1-expressing cancer cell ortumor of methods and compositions of the present invention is anon-small cell lung cancer (NSCLC) cell or tumor. In another embodiment,the NY-ESO-1-expressing cancer cell is a lung adenocarcinoma cell ortumor. In another embodiment, the NY-ESO-1-expressing cancer cell is abronchioloalveolar carcinoma (BAC) cell or tumor. In another embodiment,the NY-ESO-1-expressing cancer cell is a cell or tumor from anadenocarcinoma with bronchioloalveolar features (AdenoBAC). In anotherembodiment, the NY-ESO-1-expressing cancer cell or tumor is from asquamous cell carcinoma of the lung. Each possibility represents aseparate embodiment of the present invention.

The NY-ESO-1 peptide of methods and compositions of the presentinvention is, in another embodiment, a peptide from an NY-ESO-1 protein,wherein the sequence of the protein is:

MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR (SEQ ID NO: 1; GenBank Accession No. NM_001327). Inanother embodiment, the NY-ESO-1 protein is a homologue of SEQ ID NO: 1.In another embodiment, the NY-ESO-1 protein is a variant of SEQ IDNO: 1. In another embodiment, the NY-ESO-1 protein is an isomer of SEQID NO: 1. In another embodiment, the NY-ESO-1 protein is a fragment ofSEQ ID NO: 1. In another embodiment, the NY-ESO-1 protein is a fragmentof a homologue of SEQ ID NO: 1. In another embodiment, the NY-ESO-1protein is a fragment of a variant of SEQ ID NO: 1. In anotherembodiment, the NY-ESO-1 protein is a fragment of an isomer of SEQ IDNO: 1. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the NY-ESO-1 peptide of methods and compositionsof the present invention is derived from any other NY-ESO-1 proteinknown in the art. Each possibility represents another embodiment of thepresent invention. In another embodiment, the NY-ESO-1 antigen is apeptide having the sequence: SLLMWITQC (SEQ ID NO: 2). In anotherembodiment, the sequence is SLLMWITQCFL (SEQ ID NO: 3). In anotherembodiment, the sequence is SLLMWITQCFLP (SEQ ID NO: 4). In anotherembodiment, the sequence is SLLMWITQCFLPV (SEQ ID NO: 5). In anotherembodiment, the sequence is SLLMWITQCFLPVF (SEQ ID NO: 6). In anotherembodiment, the sequence is SLLMWITQCFLPVFL (SEQ ID NO: 7). In anotherembodiment, the sequence is WITQCFLPVFLAQPPSGQRR (SEQ ID NO: 8). Inanother embodiment, the sequence is YLAMPFATPMEAELARRSLA (SEQ ID NO: 9).In another embodiment, the sequence is ASGPGGGAPR (SEQ ID NO: 10). Inanother embodiment, the sequence is MPFATPMEA (SEQ ID NO: 11). Inanother embodiment, the sequence is LAMPFATPM (SEQ ID NO: 12). Inanother embodiment, the sequence is ARGPESRLL (SEQ ID NO: 13). Inanother embodiment, the sequence is LLMWITQCF (SEQ ID NO: 14). Inanother embodiment, the sequence is SLLMWITQV (SEQ ID NO: 15). In oneembodiment, the NY-ESO-1 antigen is a peptide comprising positions157-165 of the wild-type the NY-ESO-1 peptide. In another embodiment,the NY-ESO-1 antigen is a peptide comprising positions 53-62 of thewild-type the NY-ESO-1 peptide. In another embodiment, the NY-ESO-1antigen is a peptide comprising positions 94-102 of the wild-type theNY-ESO-1 peptide. In another embodiment, the NY-ESO-1 antigen is apeptide comprising positions 92-100 of the wild-type the NY-ESO-1peptide. In another embodiment, the NY-ESO-1 antigen is a peptidecomprising positions 80-88 of the wild-type the NY-ESO-1 peptide. Inanother embodiment, the NY-ESO-1 antigen is a peptide comprisingpositions 158-166 of the wild-type the NY-ESO-1 peptide. In anotherembodiment, the NY-ESO-1 antigen is a variant of a wild-type NY-ESO-1peptide. An example of a variant is SLLMWITQV (SEQ ID NO: 16). Eachpossibility represents another embodiment of the present invention.

In another embodiment, the antigenic peptide of interest of methods andcompositions of the present invention is a Human Papilloma Virus (HPV)E7 peptide. In another embodiment, the antigenic peptide is a whole E7protein. In another embodiment, the antigenic peptide is a fragment ofan E7 protein. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a recombinantnucleotide molecule encoding an E7-containing peptide of the presentinvention.

In another embodiment, the present invention provides a compositioncomprising a mutant-LLO containing recombinant peptide, protein orpolypeptide of the present invention and an E7 peptide.

In another embodiment, the present invention provides a recombinantvaccine vector encoding an E7-containing peptide of the presentinvention.

In another embodiment, the present invention provides a recombinantListeria strain encoding an E7-containing peptide of the presentinvention.

In another embodiment, the present invention provides a method forinducing an immune response in a subject against an HPV E7 epitope, themethod comprising the step of administering to the subject therecombinant peptide, protein or polypeptide of the present invention,thereby inducing an immune response against an HPV E7 epitope.

In one embodiment, an HPV E7 epitope for use in the compositions andmethods of the present invention is TLHEYMLDL: 7-15 (B8), YMLDLQPETT:11-20 (A2), LLMGTLGIV: 82-90 (A2), TLGIVCPI: 86-93 (A2), or another HPVE7 epitope known in the art.

In another embodiment, the present invention provides a method forinducing an immune response in a subject against an HPV E7 antigen, themethod comprising the step of administering to the subject an HPV-E7containing recombinant peptide, protein or polypeptide of the presentinvention, thereby inducing an immune response against an HPV E7antigen.

In another embodiment, the present invention provides a method forinducing an immune response in a subject against an HPV E7-expressingtarget cell, the method comprising the step of administering to thesubject an HPV-E7 containing recombinant peptide, protein or polypeptideof the present invention, thereby inducing an immune response against anHPV E7-expressing target cell. In another embodiment, the target cell isa cervical cancer cell. In another embodiment, the target cell is ahead-and-neck cancer cell. In another embodiment, the target cell is anyother type of HPV E7-expressing cell known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method fortreating an HPV E7-expressing tumor in a subject, the method comprisingthe step of administering to the subject an HPV-E7 containingrecombinant peptide, protein or polypeptide of the present invention,thereby treating an HPV E7-expressing tumor in a subject. In anotherembodiment, the tumor is a cervical tumor. In another embodiment, thetumor is a head-and-neck tumor. In another embodiment, the tumor is anyother type of HPV E7-expressing tumor known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forinhibiting or suppressing an HPV E7-expressing tumor in a subject, themethod comprising the step of administering to the subject an HPV-E7containing recombinant peptide, protein or polypeptide of the presentinvention, thereby inhibiting or suppressing an HPV E7-expressing tumorin a subject. In another embodiment, the tumor is a cervical tumor. Inanother embodiment, the tumor is a head-and-neck tumor. In anotherembodiment, the tumor is any other type of HPV E7-expressing tumor knownin the art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method forinducing regression of an HPV E7-expressing tumor in a subject, themethod comprising the step of administering to the subject an HPV-E7containing recombinant peptide, protein or polypeptide of the presentinvention, thereby inducing regression of an HPV E7-expressing tumor ina subject. In another embodiment, the tumor is a cervical tumor. Inanother embodiment, the tumor is a head-and-neck tumor. In anotherembodiment, the tumor is any other type of HPV E7-expressing tumor knownin the art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method forreducing an incidence of an HPV E7-expressing tumor in a subject, themethod comprising the step of administering to the subject an HPV-E7containing recombinant peptide, protein or polypeptide of the presentinvention, thereby reducing an incidence of an HPV E7-expressing tumorin a subject. In another embodiment, the tumor is a cervical tumor. Inanother embodiment, the tumor is a head-and-neck tumor. In anotherembodiment, the tumor is any other type of HPV E7-expressing tumor knownin the art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method forprotecting a subject against an HPV E7-expressing tumor in a subject,the method comprising the step of administering to the subject an HPV-E7containing recombinant peptide, protein or polypeptide of the presentinvention, thereby protecting a subject against an HPV E7-expressingtumor in a subject. In another embodiment, the tumor is a cervicaltumor. In another embodiment, the tumor is a head-and-neck tumor. Inanother embodiment, the tumor is any other type of HPV E7-expressingtumor known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method forinducing an immune response in a subject against an HPV E7-expressingtarget cell, the method comprising the step of administering to thesubject a vaccine vector encoding an HPV E7-containing recombinantpeptide, protein or polypeptide of the present invention, therebyinducing an immune response against an HPV E7-expressing target cell. Inanother embodiment, the target cell is a cervical cancer cell. Inanother embodiment, the target cell is a head-and-neck cancer cell. Inanother embodiment, the target cell is any other type of HPVE7-expressing cell known in the art. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the present invention provides a method fortreating or reducing an incidence of an HPV E7-expressing tumor in asubject, the method comprising the step of administering to the subjectan HPV-E7 encoding vaccine vector of the present invention, therebytreating or reducing an incidence of an HPV E7-expressing tumor in asubject. In another embodiment, the tumor is a cervical tumor. Inanother embodiment, the tumor is a head-and-neck tumor. In anotherembodiment, the tumor is any other type of HPV E7-expressing tumor knownin the art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method forinhibiting or suppressing an HPV E7-expressing tumor in a subject, themethod comprising the step of administering to the subject an HPV-E7encoding vaccine vector of the present invention, thereby inhibiting orsuppressing an HPV E7-expressing tumor in a subject. In anotherembodiment, the tumor is a cervical tumor. In another embodiment, thetumor is a head-and-neck tumor. In another embodiment, the tumor is anyother type of HPV E7-expressing tumor known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forinducing regression of an HPV E7-expressing tumor in a subject, themethod comprising the step of administering to the subject an HPV-E7encoding vaccine vector of the present invention, thereby inducingregression of an HPV E7-expressing tumor in a subject. In anotherembodiment, the tumor is a cervical tumor. In another embodiment, thetumor is a head-and-neck tumor. In another embodiment, the tumor is anyother type of HPV E7-expressing tumor known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forprotecting a subject against an HPV E7-expressing tumor in a subject,the method comprising the step of administering to the subject an HPV-E7encoding vaccine vector of the present invention, thereby protecting asubject against an HPV E7-expressing tumor in a subject. In anotherembodiment, the tumor is a cervical tumor. In another embodiment, thetumor is a head-and-neck tumor. In another embodiment, the tumor is anyother type of HPV E7-expressing tumor known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forinducing an immune response in a subject against an HPV E7-expressingtarget cell, the method comprising the step of administering to thesubject an HPV-E7 encoding nucleotide molecule of the present invention,thereby inducing an immune response against an HPV E7-expressing targetcell. In another embodiment, the target cell is a cervical cancer cell.In another embodiment, the target cell is a head-and-neck cancer cell.In another embodiment, the target cell is any other type of HPVE7-expressing cell known in the art. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the present invention provides a method fortreating or reducing an incidence of an HPV E7-expressing tumor in asubject, the method comprising the step of administering to the subjectan HPV-E7 encoding nucleotide molecule of the present invention, therebytreating or reducing an incidence of an HPV E7-expressing tumor in asubject. In another embodiment, the tumor is a cervical tumor. Inanother embodiment, the tumor is a head-and-neck tumor. In anotherembodiment, the tumor is any other type of HPV E7-expressing tumor knownin the art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method forinhibiting or suppressing an HPV E7-expressing tumor in a subject, themethod comprising the step of administering to the subject an HPV-E7encoding nucleotide molecule of the present invention, therebyinhibiting or suppressing an HPV E7-expressing tumor in a subject. Inanother embodiment, the tumor is a cervical tumor. In anotherembodiment, the tumor is a head-and-neck tumor. In another embodiment,the tumor is any other type of HPV E7-expressing tumor known in the art.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a method forinducing regression of an HPV E7-expressing tumor in a subject, themethod comprising the step of administering to the subject an HPV-E7encoding nucleotide molecule of the present invention, thereby inducingregression of an HPV E7-expressing tumor in a subject. In anotherembodiment, the tumor is a cervical tumor. In another embodiment, thetumor is a head-and-neck tumor. In another embodiment, the tumor is anyother type of HPV E7-expressing tumor known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forprotecting a subject against an HPV E7-expressing tumor in a subject,the method comprising the step of administering to the subject an HPV-E7encoding nucleotide molecule of the present invention, therebyprotecting a subject against an HPV E7-expressing tumor in a subject. Inanother embodiment, the tumor is a cervical tumor. In anotherembodiment, the tumor is a head-and-neck tumor. In another embodiment,the tumor is any other type of HPV E7-expressing tumor known in the art.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a method forinducing an immune response in a subject against an HPV E7-expressingtarget cell, the method comprising the step of administering to thesubject an a Listeria strain encoding an HPV-E7-containing peptide ofthe present invention, thereby inducing an immune response against anHPV E7-expressing target cell. In another embodiment, the target cell isa cervical cancer cell. In another embodiment, the target cell is ahead-and-neck cancer cell. In another embodiment, the target cell is anyother type of HPV E7-expressing cell known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method fortreating or reducing an incidence of an HPV E7-expressing tumor in asubject, the method comprising the step of administering to the subjectan HPV-E7 encoding Listeria strain of the present invention, therebytreating or reducing an incidence of an HPV E7-expressing tumor in asubject. In another embodiment, the tumor is a cervical tumor. Inanother embodiment, the tumor is a head-and-neck tumor. In anotherembodiment, the tumor is any other type of HPV E7-expressing tumor knownin the art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method forinhibiting or suppressing an HPV E7-expressing tumor in a subject, themethod comprising the step of administering to the subject an HPV-E7encoding Listeria strain of the present invention, thereby inhibiting orsuppressing an HPV E7-expressing tumor in a subject. In anotherembodiment, the tumor is a cervical tumor. In another embodiment, thetumor is a head-and-neck tumor. In another embodiment, the tumor is anyother type of HPV E7-expressing tumor known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forinducing regression of an HPV E7-expressing tumor in a subject, themethod comprising the step of administering to the subject an HPV-E7encoding Listeria strain of the present invention, thereby inducingregression of an HPV E7-expressing tumor in a subject. In anotherembodiment, the tumor is a cervical tumor. In another embodiment, thetumor is a head-and-neck tumor. In another embodiment, the tumor is anyother type of HPV E7-expressing tumor known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method forprotecting a subject against an HPV E7-expressing tumor in a subject,the method comprising the step of administering to the subject an HPV-E7encoding Listeria strain of the present invention, thereby protecting asubject against an HPV E7-expressing tumor in a subject. In anotherembodiment, the tumor is a cervical tumor. In another embodiment, thetumor is a head-and-neck tumor. In another embodiment, the tumor is anyother type of HPV E7-expressing tumor known in the art. Each possibilityrepresents a separate embodiment of the present invention.

The cervical tumor targeted by methods of the present invention is, inanother embodiment, a squamous cell carcinoma. In another embodiment,the cervical tumor is an adenocarcinoma. In another embodiment, thecervical tumor is an adenosquamous carcinoma. In another embodiment, thecervical tumor is a small cell carcinoma. In another embodiment, thecervical tumor is any other type of cervical tumor known in the art.Each possibility represents a separate embodiment of the presentinvention.

In one embodiment, the tumor targeted by methods of the presentinvention is a head and neck carcinoma. In another embodiment, the tumoris an anal carcinoma. In another embodiment, the tumor is a vulvarcarcinoma. In another embodiment, the tumor is a vaginal carcinoma. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, the methods provided herein may be used inconjunction with other methods of treating, inhibiting, or suppressingcervical cancer, including, inter alia, surgery, radiation therapy,chemotherapy, surveillance, adjuvant (additional), or a combination ofthese treatments.

In another embodiment, the methods provided herein may be used inconjunction with other methods of treating, inhibiting, or suppressinghead and neck carcinoma, including, inter alia, surgery, radiationtherapy, chemotherapy, surveillance, adjuvant (additional), or acombination of these treatments.

In another embodiment, the methods provided herein may be used inconjunction with other methods of treating, inhibiting, or suppressinganal carcinoma, including, inter alia, surgery, radiation therapy,chemotherapy, surveillance, adjuvant (additional), or a combination ofthese treatments.

In another embodiment, the methods provided herein may be used inconjunction with other methods of treating, inhibiting, or suppressingvulvar carcinoma, including, inter alia, surgery, radiation therapy,chemotherapy, surveillance, adjuvant (additional), or a combination ofthese treatments.

In another embodiment, the methods provided herein may be used inconjunction with other methods of treating, inhibiting, or suppressingvaginal carcinoma, including, inter alia, surgery, radiation therapy,chemotherapy, surveillance, adjuvant (additional), or a combination ofthese treatments.

The E7 protein that is utilized (either whole or as the source of thefragments) has, in another embodiment, the sequence

MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP(SEQ ID NO: 17). In another embodiment, the E7 protein is a homologue ofSEQ ID NO: 17. In another embodiment, the E7 protein is a variant of SEQID NO: 17. In another embodiment, the E7 protein is an isomer of SEQ IDNO: 17. In another embodiment, the E7 protein is a fragment of SEQ IDNO: 17. In another embodiment, the E7 protein is a fragment of ahomologue of SEQ ID NO: 17. In another embodiment, the E7 protein is afragment of a variant of SEQ ID NO: 17. In another embodiment, the E7protein is a fragment of an isomer of SEQ ID NO: 17. Each possibilityrepresents a separate embodiment of the present invention.

In one embodiment, the cholesterol binding domain of LLO (ECTGLAWEWWR;SEQ ID NO: 18) is substituted with an E7 epitope (RAHYNIVTF; SEQ ID NO:19).

In another embodiment, the sequence of the E7 protein is:

MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ(SEQ ID NO: 20). In another embodiment, the E7 protein is a homologue ofSEQ ID NO: 20. In another embodiment, the E7 protein is a variant of SEQID NO: 20. In another embodiment, the E7 protein is an isomer of SEQ IDNO: 20. In another embodiment, the E7 protein is a fragment of SEQ IDNO: 20. In another embodiment, the E7 protein is a fragment of ahomologue of SEQ ID NO: 20. In another embodiment, the E7 protein is afragment of a variant of SEQ ID NO: 20. In another embodiment, the E7protein is a fragment of an isomer of SEQ ID NO: 20. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the E7 protein has a sequence set forth in one ofthe following GenBank entries: M24215, NC_004500, V01116, X62843, orM14119. In another embodiment, the E7 protein is a homologue of asequence from one of the above GenBank entries. In another embodiment,the E7 protein is a variant of a sequence from one of the above GenBankentries. In another embodiment, the E7 protein is an isomer of asequence from one of the above GenBank entries. In another embodiment,the E7 protein is a fragment of a sequence from one of the above GenBankentries. In another embodiment, the E7 protein is a fragment of ahomologue of a sequence from one of the above GenBank entries. Inanother embodiment, the E7 protein is a fragment of a variant of asequence from one of the above GenBank entries. In another embodiment,the E7 protein is a fragment of an isomer of a sequence from one of theabove GenBank entries. Each possibility represents a separate embodimentof the present invention.

In one embodiment, the HPV16 E7 antigen is a peptide having thesequence: TLGIVCPI (SEQ ID NO: 21). In another embodiment, the HPV16 E7antigen is a peptide having the sequence: LLMGTLGIV (SEQ ID NO: 22). Inanother embodiment, the HPV16 E7 antigen is a peptide having thesequence: YMLDLQPETT (SEQ ID NO: 23). In one embodiment, the HPV16 E7antigen is a peptide comprising positions 86-93 of the wild-type HPV16E7 antigen. In one embodiment, the HPV16 E7 antigen is a peptidecomprising positions 82-90 of the wild-type HPV16 E7 antigen. In oneembodiment, the HPV16 E7 antigen is a peptide comprising positions 11-20of the wild-type HPV16 E7 antigen. In another embodiment, the HPV16 E7antigen is a peptide consisting of positions 86-93, 82-90, or 11-20 ofthe wild-type HPV16 E7 antigen. In another embodiment, the HPV16 E7antigen is a variant of a wild-type HPV16 E7 peptide. In anotherembodiment, the HPV16 E7 antigen is any HPV16 E7 antigen described inRessing at al., J Immunol 1995 154(11):5934-43, which is incorporatedherein by reference in its entirety.

Each possibility represents another embodiment of the present invention.

In another embodiment, the antigenic peptide of interest of methods andcompositions of the present invention is an HPV E6 peptide. In anotherembodiment, the antigenic peptide is a whole E6 protein. In anotherembodiment, the antigenic peptide is a fragment of an E6 protein. Eachpossibility represents a separate embodiment of the present invention.

The E6 protein that is utilized (either whole or as the source of thefragments) has, in another embodiment, the sequence

MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL (SEQ ID NO: 24). Inanother embodiment, the E6 protein is a homologue of SEQ ID NO: 24. Inanother embodiment, the E6 protein is a variant of SEQ ID NO: 24. Inanother embodiment, the E6 protein is an isomer of SEQ ID NO: 24. Inanother embodiment, the E6 protein is a fragment of SEQ ID NO: 24. Inanother embodiment, the E6 protein is a fragment of a homologue of SEQID NO: 24. In another embodiment, the E6 protein is a fragment of avariant of SEQ ID NO: 24. In another embodiment, the E6 protein is afragment of an isomer of SEQ ID NO: 24. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the sequence of the E6 protein is:

MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV (SEQ ID NO: 25). Inanother embodiment, In another embodiment, the E6 protein is a homologueof SEQ ID NO: 25. In another embodiment, the E6 protein is a variant ofSEQ ID NO: 25. In another embodiment, the E6 protein is an isomer of SEQID NO: 25. In another embodiment, the E6 protein is a fragment of SEQ IDNO: 25. In another embodiment, the E6 protein is a fragment of ahomologue of SEQ ID NO: 25. In another embodiment, the E6 protein is afragment of a variant of SEQ ID NO: 25. In another embodiment, the E6protein is a fragment of an isomer of SEQ ID NO: 25. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the E6 protein has a sequence set forth in one ofthe following GenBank entries: M24215, M14119, NC_004500, V01116,X62843, or M14119. In another embodiment, the E6 protein is a homologueof a sequence from one of the above GenBank entries. In anotherembodiment, the E6 protein is a variant of a sequence from one of theabove GenBank entries. In another embodiment, the E6 protein is anisomer of a sequence from one of the above GenBank entries. In anotherembodiment, the E6 protein is a fragment of a sequence from one of theabove GenBank entries. In another embodiment, the E6 protein is afragment of a homologue of a sequence from one of the above GenBankentries. In another embodiment, the E6 protein is a fragment of avariant of a sequence from one of the above GenBank entries. In anotherembodiment, the E6 protein is a fragment of an isomer of a sequence fromone of the above GenBank entries. Each possibility represents a separateembodiment of the present invention.

The HPV that is the source of the heterologous antigen of methods of thepresent invention is, in another embodiment, an HPV 16. In anotherembodiment, the HPV is an HPV-18. In another embodiment, the HPV isselected from HPV-16 and HPV-18. In another embodiment, the HPV is anHPV-31. In another embodiment, the HPV is an HPV-35. In anotherembodiment, the HPV is an HPV-39. In another embodiment, the HPV is anHPV-45. In another embodiment, the HPV is an HPV-51. In anotherembodiment, the HPV is an HPV-52. In another embodiment, the HPV is anHPV-58. In another embodiment, the HPV is a high-risk HPV type. Inanother embodiment, the HPV is a mucosal HPV type. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the antigenic peptide of interest of methods andcompositions of the present invention is a BCR idiotype.

Cytogenetic studies have shown that some histological and immunologicalsub-types of NHL have chromosomal abnormalities with reciprocaltranslocations, frequently involving genes for the B-cell receptor andan oncogene. Lymphomagenesis results in clonal expansion of thetransformed B-cell, with each daughter cell expressing the BCR on thecell surface as well as BCR-derived peptides associated with MHC class Iand II molecules. The BCR has a unique conformation formed by thehypervariable regions of the heavy and light chain, this is referred toas the “idiotype,” is the same for every daughter cell within the tumor,and is not present on significant numbers of normal cells. Therefore,the idiotype is a specific tumor antigen and a target for lymphomatherapy.

As provided herein, the present invention has produced aconformationally intact fusion protein comprising an LLO protein and aBCR idiotype (Experimental Details section herein).

In another embodiment, the present invention provides a method forinducing an immune response against a lymphoma in a subject, comprisingthe step of administering to the subject a BCR idiotype-containingrecombinant peptide, protein or polypeptide of the present invention,thereby inducing an immune response against a lymphoma.

In another embodiment, the present invention provides a method forinducing an immune response against a lymphoma in a subject, comprisingthe step of administering to the subject a BCR idiotype-encodingnucleotide molecule of the present invention, thereby inducing an immuneresponse against a lymphoma.

In another embodiment, the present invention provides a method forinducing an immune response against a lymphoma in a subject, comprisingthe step of administering to the subject a BCR idiotype-expressingrecombinant vaccine vector of the present invention, thereby inducing animmune response against a lymphoma.

In another embodiment, the present invention provides a method forinducing an immune response against a lymphoma in a subject, comprisingthe step of administering to the subject a BCR idiotype-expressingListeria strain of the present invention, thereby inducing an immuneresponse against a lymphoma.

In another embodiment, the present invention provides a method fortreating, inhibiting, or suppressing a lymphoma in a subject, comprisingthe step of administering to the subject a BCR idiotype-containingpeptide of the present invention, thereby treating a lymphoma.

In another embodiment, the present invention provides a method fortreating, inhibiting, or suppressing a lymphoma in a subject, comprisingthe step of administering to the subject a BCR idiotype-encodingnucleotide molecule of the present invention, thereby treating alymphoma.

In another embodiment, the present invention provides a method fortreating, inhibiting, or suppressing a lymphoma in a subject, comprisingthe step of administering to the subject a BCR idiotype-expressingrecombinant vaccine vector of the present invention, thereby treating alymphoma in a subject.

In another embodiment, the present invention provides a method fortreating, inhibiting, or suppressing a lymphoma in a subject, comprisingthe step of administering to the subject a BCR idiotype-expressingListeria strain of the present invention, thereby treating a lymphoma ina subject.

In another embodiment, the present invention provides a method forinducing a regression of a lymphoma in a subject, comprising the step ofadministering to the subject a BCR idiotype-containing peptide of thepresent invention, thereby inducing a regression of a lymphoma.

In another embodiment, the present invention provides a method forinducing a regression of a lymphoma in a subject, comprising the step ofadministering to the subject a BCR idiotype-encoding nucleotide moleculeof the present invention, thereby inducing a regression of a lymphoma.

In another embodiment, the present invention provides a method forinducing a regression of a lymphoma in a subject, comprising the step ofadministering to the subject a BCR idiotype-expressing recombinantvaccine vector of the present invention, thereby inducing a regressionof a lymphoma in a subject.

In another embodiment, the present invention provides a method forinducing a regression of a lymphoma in a subject, comprising the step ofadministering to the subject a BCR idiotype-expressing Listeria strainof the present invention, thereby inducing a regression of a lymphoma ina subject.

As provided in the Experimental Details section herein, fusion of LLO toan antigen increases its immunogenicity. In addition, administration offusion proteins of the present invention results in protection againsttumor challenge.

Moreover, as provided herein, the present invention has produced aconformationally intact fusion protein comprising an LLO protein and aBCR idiotype, has demonstrated accurate and effective methodologies fortesting anti-lymphoma vaccines in mouse and animal models, and has shownthe efficacy of vaccines of the present invention in protecting againstlymphoma and their superiority over currently accepted anti-lymphomavaccines (Experimental Details section).

In one embodiment, a vaccine of the present invention is a compositionthat upon administration stimulates antibody production or cellularimmunity against an antigen.

In one embodiment, vaccines are administered as killed or attenuatedmicro-organisms, while in another embodiment, vaccines comprise naturalor genetically engineered antigens. In one embodiment, effectivevaccines stimulate the immune system to promote the development ofantibodies that can quickly and effectively attack cells, microorganismsor viruses that produce the antigen against which the subject wasvaccination, when they are produced in the subject, thereby preventingdisease development.

In one embodiment, a vaccine of the present invention is prophylactic,while in another embodiment, a vaccine of the present invention istherapeutic. In one embodiment, a prophylactic vaccine is administeredto a population that is susceptible to developing or contracting aparticular disease or condition, whether via environmental exposure orgenetic predisposition. Such susceptibility factors aredisease-dependent and are well-known to those of skill in the art. Forexample, the population comprising smokers (in one embodiment,cigarette, cigar, pipe, etc) is known in the art to be susceptible todeveloping lung cancer. The population comprising a mutation in BRCA-1and BRCA-2 is known in the art to be susceptible to breast and/orovarian cancer. The population comprising particular single nucleotidepolymorphisms (SNPs) in chromosome 15 inside a region that containsgenes for the nicotinic acetylcholine receptor alpha subunits 3 and 5 isknown in the art to be susceptible to lung cancer. Other similarsusceptibility factors are known in the art, and such susceptiblepopulations are envisioned in one embodiment, to be a population forwhich a prophylactic vaccine of the instant invention would be mostuseful.

Thus, vaccines of the present invention are efficacious in inducing animmune response to, preventing, treating, and inducing remission oflymphoma. In another embodiment, the present invention provides a methodfor overcoming an immune tolerance to a lymphoma in a subject,comprising the step of administering to the subject a peptide of thepresent invention, thereby overcoming an immune tolerance to a lymphoma.

In another embodiment, the present invention provides a method forovercoming an immune tolerance to a lymphoma in a subject, comprisingthe step of administering to the subject a nucleotide molecule of thepresent invention, thereby overcoming an immune tolerance to a lymphoma.

“Tolerance” refers, in another embodiment, to a lack of responsivenessof the host to an antigen. In another embodiment, the term refers to alack of detectable responsiveness of the host to an antigen. In anotherembodiment, the term refers to a lack of immunogenicity of an antigen ina host. In another embodiment, tolerance is measured by lack ofresponsiveness in an in vitro CTL killing assay. In another embodiment,tolerance is measured by lack of responsiveness in a delayed-typehypersensitivity assay. In another embodiment, tolerance is measured bylack of responsiveness in any other suitable assay known in the art. Inanother embodiment, tolerance is determined or measured as depicted inthe Examples herein. Each possibility represents another embodiment ofthe present invention.

“Overcome” refers, in another embodiment, to a reversal of tolerance bya vaccine. In another embodiment, the term refers to conferment ofdetectable immune response by a vaccine. In another embodiment,overcoming of immune tolerance is determined or measured as depicted inthe Examples herein. Each possibility represents another embodiment ofthe present invention.

In another embodiment, the present invention provides a method forreducing an incidence of relapse of a lymphoma in a subject in remissionfrom the lymphoma, comprising the step of administering to the subject apeptide of the present invention, thereby reducing an incidence ofrelapse of a lymphoma in a subject in remission from the lymphoma.

In another embodiment, the present invention provides a method forreducing an incidence of relapse of a lymphoma in a subject in remissionfrom the lymphoma, comprising administering to the subject a nucleotidemolecule of the present invention, thereby reducing an incidence ofrelapse of a lymphoma in a subject in remission from the lymphoma.

In another embodiment, the present invention provides a method forsuppressing a formation of a lymphoma, comprising the step ofadministering a recombinant peptide, protein or polypeptide of thepresent invention thereby suppressing a formation of a lymphoma.

In another embodiment, the present invention provides a method forsuppressing a formation of a lymphoma, comprising the step ofadministering a nucleotide molecule of the present invention, therebysuppressing a formation of a lymphoma.

In another embodiment, the present invention provides a method ofinducing a remission of a residual B cell lymphoma disease, comprisingadministering a peptide of the present invention, thereby inducing aremission of a residual B cell lymphoma disease.

In another embodiment, the present invention provides a method ofinducing a remission of a residual B cell lymphoma disease, comprisingadministering a nucleotide molecule of the present invention, therebyinducing a remission of a residual B cell lymphoma disease.

In another embodiment, the present invention provides a method ofeliminating minimal residual B cell lymphoma disease, comprisingadministering a peptide of the present invention, thereby eliminatingminimal residual B cell lymphoma disease.

In another embodiment, the present invention provides a method ofeliminating minimal residual B cell lymphoma disease, comprisingadministering a nucleotide molecule of the present invention, therebyeliminating minimal residual B cell lymphoma disease.

In another embodiment, the present invention provides a method ofreducing a size of a B cell lymphoma, comprising administering a peptideof the present invention, thereby reducing a size of a B cell lymphoma.

In another embodiment, the present invention provides a method ofreducing a size of a B cell lymphoma, comprising administering anucleotide molecule of the present invention, thereby reducing a size ofa B cell lymphoma.

In another embodiment, the present invention provides a method ofreducing a volume of a B cell lymphoma, comprising administering apeptide of the present invention, thereby reducing a volume of a B celllymphoma.

In another embodiment, the present invention provides a method ofreducing a volume of a B cell lymphoma, comprising administering anucleotide molecule of the present invention, thereby reducing a volumeof a B cell lymphoma.

In another embodiment, the lymphoma that is a target of a method ofpresent invention is, in another embodiment, a Non-Hodgkin's Lymphoma.In another embodiment, a lymphoma is a B cell lymphoma. In anotherembodiment, a lymphoma is a low-grade lymphoma. In another embodiment, alymphoma is a low-grade NHL. In another embodiment, a lymphoma isresidual disease from one of the above types of lymphoma. In anotherembodiment, the lymphoma is any other type of lymphoma known in the art.In another embodiment, the lymphoma is a Burkitt's Lymphoma. In anotherembodiment, the lymphoma is follicular lymphoma. In another embodiment,the lymphoma is marginal zone lymphoma. In another embodiment, thelymphoma is splenic marginal zone lymphoma. In another embodiment, thelymphoma is a mantle cell lymphoma. In another embodiment, the lymphomais an indolent mantle cell lymphoma. In another embodiment, the lymphomais any other known type of lymphoma that expresses a BCR. Each type oflymphoma represents a separate embodiment of the present invention.

In another embodiment, cells of the tumor that is targeted by methodsand compositions of the present invention express a BCR. In anotherembodiment, the tumor is associated with a BCR. In another embodiment,the BCR has an idiotype that is characteristic of the tumor. In anotherembodiment, the BCR expressed by a tumor cell is the target of theimmune responses induced by methods and compositions of the presentinvention.

In another embodiment, the BCR expressed by the target cell is requiredfor a tumor phenotype. In another embodiment, the BCR is necessary fortransformation of a tumor cell. In another embodiment, tumor cells thatlose expression of the BCR lose their uncontrolled growth, invasiveness,or another feature of malignancy. Each possibility represents a separateembodiment of the present invention.

Methods and compositions of the present invention apply equally to anyBCR of a non-Hodgkin's lymphoma and any idiotype thereof. Sequences ofBCR are well known in the art, and are readily obtained from lymphomasamples.

An exemplary sequence of a BCR immunoglobulin (Ig) heavy chain precursoris:

(SEQ ID NO: 26; GenBank Accession No. X14096)MKLWLNWIFLVTLLNGIQCEVKLVESGGGLVQPGGSLSLSCAASGFTFTDYYMSWVRQPPGKALEWLALIRNKANGYTTEYSASVKGRFTISRDNSQSILYLQMNALRAEDSATYYCARDPNYYDGSYEGYFDYWAQGTTLTVSS.

An exemplary sequence of a BCR Ig light chain precursor is:

(SEQ ID NO: 27; GenBank Accession No. X14097)LLLISVTVIVSNGEIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQKPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSIPRGVTFGSGTKLEIKR.

Another exemplary sequence of a BCR Ig light chain precursor is:

(SEQ ID NO: 28; GenBank Accession No. X14098)GFLLISVTVILTNGEIFLTQSPAIIAASPGEKVTITCSASSSVSYMNWYQQKPGSSPKIWIYGISNLASGVPARFSGSGSGTSFSFTINSMEAEDVATYYCQQRSSYPFTFGSGTKLEIKRADAAPTVSHLP.

Another exemplary sequence of a BCR Ig light chain precursor is:

(SEQ ID NO: 29; GenBank Accession No. X14099)LLLISVTVIVSNGEIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQKPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSIPRTFGSGTKLEIKRA.

Another exemplary sequence of a BCR Ig heavy chain is:

(SEQ ID NO: 30; human follicular lymphoma IgMheavy chain; GenBank Accession No. X70200)MEFGLSWVFLVAILKGVQCEMQLVESGGGLVQPGESLKLSCAASGFSFSGSTIHWVRQASGRGLEWVGRSRSKADNFMTSYAPSIKGKFIISRDDSSNMLYLQMNNLKTEDTAVYFCTRNFTSLDSTGNSFGPWGQGTLVTVSSGSASAP TLFPLVS.

Another exemplary sequence of a BCR Ig heavy chain is:

(SEQ ID NO: 31; human follicular lymphoma IgMheavy chain; GenBank Accession No. X70199)MEFGLSWVFLVAILKGVQCEMQLVESGGGLVQPGESFKLSCAASGFSFSGSTIHWVRQASGRGLEWVGRSRSKADNFMTSYAPSIKGKFIISRDDSSNMMYLQMNNLKNEDTAVYFCTRNFTSLDSTGNSFGPWGQGTLVTVSSGSASAP TLFPLVS.

Another exemplary sequence of a BCR Ig heavy chain is:

(SEQ ID NO: 32; human follicular lymphoma IgMheavy chain; GenBank Accession No. X70208)MEFGLSWVFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHTVRGGHCAPRHKPSLQERWGNQRQGALRS.

Another exemplary sequence of a BCR Ig heavy chain is:

(SEQ ID NO: 33; human follicular lymphoma IgMheavy chain; GenBank Accession No. X70207)MEFGLSWVFLVAILKGVQCEVQLVESGGGLVQPGGSLKLSCAASGFTFSGSAMHWVRQASGKGLEWVGHIRDKANSYATTYAASVKGRFTISRDDSKNTAYLQMNSLKIEDTAVYFCTRNFTSLDSTGNSFGPW.

Another exemplary sequence of a BCR Ig light chain is:

(SEQ ID NO: 34; human lymphoplasmacytic/lympho-plasmacytoid immunocytoma light chain; GenBank Accession No. AAD14088)SELTQDPVVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNLPLFGG GTKLTVLG.

Another exemplary sequence of a BCR Ig light chain is:

(SEQ ID NO: 35; human follicular lymphoma lightchain; GenBank Accession No. Y09250)DIQMTQSPDSLTVSLGERATINCKSSQSILYSSNDKNYLAWYQQKAGQPPKLLIYWASTRESGVPDRFSGSGSATDFTLTISSLQAEDVAIYYCQQYYST PLTFGGGTKVEIKR.

Another exemplary sequence of a BCR Ig light chain is:

(SEQ ID NO: 36; human splenic marginal zone lym- phoma light chain; GenBank Accession No. AAX93805)DIQMTQSPSTLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFVTYYCQQYNTFSSYTFG QGTKVEIK.

Sequences of other exemplary BCR Ig light chains are found in GenBankAccession Nos. AAX93769-93802, CAA25477, AAB31509, CAE52829-CAE52832,AAF79132-79143, and other sequences found in GenBank. Each sequencerepresents a separate embodiment of the present invention. CAA73059

Sequences of other exemplary BCR Ig heavy chains are found in GenBankAccession Nos. CAA73044-73059, AAX93809-AAX93842, AAQ74129, CAC39369,AAB52590-AAB52597, and other sequences found in GenBank. Each sequencerepresents a separate embodiment of the present invention.

Methods for determining complementarity-determining regions (cdr) of aBCR are well known in the art. For example, the CDR1 of SEQ ID NO: 26consists of residues 50-54; the CDR2 consists of residues 66-87; the Dsegment consists of residues 120-130; and the J segment consists ofresidues 131-145. The CDR1 of SEQ ID NO: 30 consists of residues148-162; the FR2 consists of residues 163-204; the CDR2 consists ofresidues 205-261; the FR3 consists of residues 262-357; the CDR3consists of residues 358-432; and the CH1 consists of residues 433-473.In another embodiment, the framework regions (non-cdr regions) aredetermined by homology with known framework regions of otherimmunoglobulin molecules from the same species. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, an idiotype is identified by determining the cdrof a BCR of methods and compositions of the present invention.

In another embodiment, a complete BCR is contained or utilized inmethods and compositions of the present invention. In anotherembodiment, a fragment of a BCR is contained or utilized. In anotherembodiment, the BCR fragment contains the idiotype thereof. In anotherembodiment, the BCR fragment contains a T cell epitope. In anotherembodiment, the BCR fragment contains an antibody epitope. In anotherembodiment, “antigen” is used herein to refer to the BCR or fragmentthereof that is the target of immune responses induced by methods andcompositions of the present invention.

In another embodiment, the fragment of a BCR contained in peptides ofthe present invention is a single chain fragment of the variable regions(scFV) of the BCR. In another embodiment, the BCR fragment isconformationally intact. In another embodiment, the BCR fragmentcontains the idiotype of the BCR. In another embodiment, the BCRidiotype is conformationally intact. Each possibility represents aseparate embodiment of the present invention.

“Idiotype” refers, in another embodiment, to the structure formed by thecomplementarity-determining region (cdr) of a BCR. In anotherembodiment, the term refers to the unique region of a BCR. In anotherembodiment, the term refers to the antigen-binding site of the BCR. Eachpossibility represents a separate embodiment of the present invention.

“Conformationally intact” refers, in another embodiment, to aconformation that is not significantly altered relative to the nativeconformation. In another embodiment, the term refers to an antibodyreactivity that is not significantly altered relative to the nativeprotein. In another embodiment, the term refers to an antibodyreactivity that overlaps substantially with the native protein. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, a peptide utilized in methods of the presentinvention comprises an idiotype that is homologous to an idiotypeexpressed by cells of the lymphoma. In another embodiment, the peptidecomprises an idiotype that is identical to an idiotype expressed bycells of the lymphoma. Each possibility represents a separate embodimentof the present invention.

In another embodiment, a nucleotide molecule utilized in methods of thepresent invention encodes an idiotype that is homologous to an idiotypeexpressed by cells of the lymphoma. In another embodiment, thenucleotide molecule encodes an idiotype that is identical to an idiotypeexpressed by cells of the lymphoma. In another embodiment, the antigenis highly homologous to the antigen expressed by the tumor cell. “Highlyhomologous” refers, in another embodiment, to a homology of greater than90%. In another embodiment, the term refers to a homology of greaterthan 92%. In another embodiment, the term refers to a homology ofgreater than 93%. In another embodiment, the term refers to a homologyof greater than 94%. In another embodiment, the term refers to ahomology of greater than 95%. In another embodiment, the term refers toa homology of greater than 96%. In another embodiment, the term refersto a homology of greater than 97%. In another embodiment, the termrefers to a homology of greater than 98%. In another embodiment, theterm refers to a homology of greater than 99%. In another embodiment,the term refers to a homology of 100%. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the residual B cell lymphoma disease or minimalresidual B cell lymphoma disease treated by a method of the presentinvention is that remaining after de-bulking therapy. Methods forperforming de-bulking therapy are well known in the art, and aredescribed, for example, in Winter J N et al (Low-grade lymphoma.Hematology (Am Soc Hematol Educ Program). 2004; 203-20) and Buske C etal (Current status and perspective of antibody therapy in follicularlymphoma. Haematologica. 2006 January; 91(1):104-12). Each possibilityrepresents a separate embodiment of the present invention.

The heterologous antigenic peptide of methods and compositions of thepresent invention is, in another embodiment, an antigenic protein. Inanother embodiment, the antigenic peptide is a fragment of an antigenicprotein. In another embodiment, the antigenic peptide is an immunogenicpeptide derived from tumor. In another embodiment, the antigenic peptideis an immunogenic peptide derived from metastasis. In anotherembodiment, the antigenic peptide is an immunogenic peptide derived fromcancerous cells. In another embodiment, the antigenic peptide is apro-angiogenesis immunogenic peptide.

In another embodiment, the antigenic polypeptide is Human PapillomaVirus-E7 (HPV-E7) antigen, which in one embodiment, is from HPV16 (inone embodiment, GenBank Accession No. AAD33253) and in anotherembodiment, from HPV18 (in one embodiment, GenBank Accession No.P06788). In another embodiment, the antigenic polypeptide is HPV-E6,which in one embodiment, is from HPV16 (in one embodiment, GenBankAccession No. AAD33252, AAM51854, AAM51853, or AAB67615) and in anotherembodiment, from HPV18 (in one embodiment, GenBank Accession No.P06463). In another embodiment, the antigenic polypeptide is a Her/2-neuantigen. In another embodiment, the antigenic polypeptide is ProstateSpecific Antigen (PSA) (in one embodiment, GenBank Accession No.CAD30844, CAD54617, AAA58802, or NP_001639). In another embodiment, theantigenic polypeptide is Stratum Corneum Chymotryptic Enzyme (SCCE)antigen (in one embodiment, GenBank Accession No. AAK69652, AAK69624,AAG33360, AAF01139, or AAC37551). In another embodiment, the antigenicpolypeptide is Wilms tumor antigen 1, which in another embodiment isWT-1 Telomerase (GenBank Accession. No. P49952, P22561, NP_659032,CAC39220.2, or EAW68222.1). In another embodiment, the antigenicpolypeptide is hTERT or Telomerase (GenBank Accession. No. NM003219(variant 1), NM198255 (variant 2), NM 198253 (variant 3), or NM 198254(variant 4). In another embodiment, the antigenic polypeptide isProteinase 3 (in one embodiment, GenBank Accession No. M29142, M75154,M96839, X55668, NM 00277, M96628 or X56606). In another embodiment, theantigenic polypeptide is Tyrosinase Related Protein 2 (TRP2) (in oneembodiment, GenBank Accession No. NP_001913, ABI73976, AAP33051, orQ95119). In another embodiment, the antigenic polypeptide is HighMolecular Weight Melanoma Associated Antigen (HMW-MAA) (in oneembodiment, GenBank Accession No. NP_001888, AAI28111, or AAQ62842). Inanother embodiment, the antigenic polypeptide is Testisin (in oneembodiment, GenBank Accession No. AAF79020, AAF79019, AAG02255,AAK29360, AAD41588, or NP_659206). In another embodiment, the antigenicpolypeptide is NY-ESO-1 antigen (in one embodiment, GenBank AccessionNo. CAA05908, P78358, AAB49693, or NP_640343). In another embodiment,the antigenic polypeptide is PSCA (in one embodiment, GenBank AccessionNo. AAH65183, NP_005663, NP_082492, 043653, orCAB97347). In anotherembodiment, the antigenic polypeptide is Interleukin (IL) 13 Receptoralpha (in one embodiment, GenBank Accession No. NP_000631, NP_001551,NP_032382, NP_598751, NP_001003075, or NP_999506). In anotherembodiment, the antigenic polypeptide is Carbonic anhydrase IX (CAIX)(in one embodiment, GenBank Accession No. CAI13455, CAI10985, EAW58359,NP_001207, NP_647466, or NP_001101426). In another embodiment, theantigenic polypeptide is carcinoembryonic antigen (CEA) (in oneembodiment, GenBank Accession No. AAA66186, CAA79884, CAA66955,AAA51966, AAD15250, or AAA51970.). In another embodiment, the antigenicpolypeptide is MAGE-A (in one embodiment, GenBank Accession No.NP_786885, NP_786884, NP_005352, NP_004979, NP_005358, or NP_005353). Inanother embodiment, the antigenic polypeptide is survivin (in oneembodiment, GenBank Accession No. AAC51660, AAY15202, ABF60110,NP_001003019, or NP_001082350). In another embodiment, the antigenicpolypeptide is GP100 (in one embodiment, GenBank Accession No. AAC60634,YP_655861, or AAB31176). In another embodiment, the antigenicpolypeptide is any other antigenic polypeptide known in the art. Inanother embodiment, the antigenic peptide of the compositions andmethods of the present invention comprise an immunogenic portion of theantigenic polypeptide. Each possibility represents a separate embodimentof the present invention.

In other embodiments, the antigen is derived from a fungal pathogen,bacteria, parasite, helminth, or viruses. In other embodiments, theantigen is selected from tetanus toxoid, hemagglutinin molecules frominfluenza virus, diphtheria toxoid, HIV gp120, HIV gag protein, HIV envprotein, IgA protease, insulin peptide B, Spongospora subterraneaantigen, vibriose antigens, Salmonella antigens, pneumococcus antigens,respiratory syncytial virus antigens, Haemophilus influenza outermembrane proteins, Helicobacter pylori urease, Neisseria meningitidispilins, N. gonorrhoeae pilins, human papilloma virus antigens E1 and E2from type HPV-16, -18, -31, -33, -35 or -45 human papilloma viruses, thetumor antigens CEA, the ras protein, mutated or otherwise, the p53protein, mutated or otherwise.

In various embodiments, the antigen of methods and compositions of thepresent invention includes but is not limited to antigens from thefollowing infectious diseases, measles, mumps, rubella, poliomyelitis,hepatitis A, B (e.g., GenBank Accession No. E02707), and C (e.g.,GenBank Accession No. E06890), as well as other hepatitis viruses, typeA influenza, other types of influenza, adenovirus (e.g., types 4 and 7),rabies (e.g., GenBank Accession No. M34678), yellow fever, Japaneseencephalitis (e.g., GenBank Accession No. E07883), dengue (e.g., GenBankAccession No. M24444), hantavirus, and HIV (e.g., GenBank Accession No.U18552). Bacterial and parasitic antigens will be derived from knowncausative agents responsible for diseases including, but not limited to,diphtheria, pertussis (e.g., GenBank Accession No. M35274), tetanus(e.g., GenBank Accession No. M64353), tuberculosis, bacterial and fungalpneumonias (e.g., Haemophilus influenzae, Pneumocystis carinii, etc.),cholera, typhoid, plague, shigellosis, salmonellosis (e.g., GenBankAccession No. L03833), Legionnaire's Disease, Lyme disease (e.g.,GenBank Accession No. U59487), malaria (e.g., GenBank Accession No.X53832), hookworm, onchocerciasis (e.g., GenBank Accession No. M27807),schistosomiasis (e.g., GenBank Accession No. L08198), trypanosomiasis,leshmaniasis, giardiasis (e.g., GenBank Accession No. M33641),amoebiasis, filariasis (e.g., GenBank Accession No. J03266),borreliosis, and trichinosis.

In other embodiments, the antigen is one of the following tumorantigens: any of the various MAGEs (Melanoma-Associated Antigen E),including MAGE 1 (e.g., GenBank Accession No. M77481), MAGE 2 (e.g.,GenBank Accession No. U03735), MAGE 3, MAGE 4, TRP-2, gp-100,tyrosinase, MART-1, HSP-70, and beta-HCG; a tyrosinase; mutant ras;mutant p53 (e.g., GenBank Accession No. X54156 and AA494311); and p97melanoma antigen (e.g., GenBank Accession No. M12154). Othertumor-specific antigens include the Ras peptide and p53 peptideassociated with advanced cancers, the HPV 16/18 and E6/E7 antigensassociated with cervical cancers, MUC1 antigen associated with breastcarcinoma (e.g., GenBank Accession No. J0365 1), CEA (carcinoembryonicantigen) associated with colorectal cancer (e.g., GenBank Accession No.X983 11), gp100 (e.g., GenBank Accession No. 573003) or MART1 antigensassociated with melanoma, and the prostate-specific antigen (KLK3)associated with prostate cancer (e.g., GenBank Accession No. X14810).The p53 gene sequence is known (See e.g., Harris et al. (1986) Mol.Cell. Biol., 6:4650-4656) and is deposited with GenBank under AccessionNo. M14694. Tumor antigens encompassed by the present invention furtherinclude, but are not limited to, Her-2/Neu (e.g. GenBank Accession Nos.M16789.1, M16790.1, M16791.1, M16792.1), NY-ESO-1 (e.g. GenBankAccession No. U87459), WT-1 (e.g. GenBank Accession Nos. NM000378(variant A), NM024424 (variant B), NM 024425 (variant C), and NM024426(variant D)), LAGE-1 (e.g. GenBank Accession No. CAA11044), synovialsarcoma, X (SSX)-2; (e.g. GenBank Accession No. NP_003138, NP_783629,NP_783729, NP_066295), and stratum corneum chymotryptic enzyme (SCCE;GenBank Accession No. NM_005046 and NM_139277)). Thus, the presentinvention can be used as immunotherapeutics for cancers including, butnot limited to, cervical, breast, colorectal, prostate, lung cancers,and for melanomas.

Each antigen represents a separate embodiment of the present invention.

In one embodiment, methods of evaluating the production of an immuneresponse by a subject to an antigen are known in the art, and in oneembodiment, are described hereinbelow in the Examples section.

The LLO protein utilized to construct vaccines of the present invention(in another embodiment, used as the source of the LLO fragmentincorporated in the vaccines) has, in another embodiment, the sequence:

MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIE (GenBank Accession No. P13128; SEQ ID NO: 37; nucleicacid sequence is set forth in GenBank Accession No. X15127). The first25 AA of the proprotein corresponding to this sequence are the signalsequence and are cleaved from LLO when it is secreted by the bacterium.Thus, in this embodiment, the full-length active LLO protein is 504residues long. In another embodiment, the LLO protein is a homologue ofSEQ ID NO: 37. In another embodiment, the LLO protein is a variant ofSEQ ID NO: 37. In another embodiment, the LLO protein is an isomer ofSEQ ID NO: 37. In another embodiment, the LLO protein is a fragment ofSEQ ID NO: 37. In another embodiment, the LLO protein is a fragment of ahomologue of SEQ ID NO: 37. In another embodiment, the LLO protein is afragment of a variant of SEQ ID NO: 37. In another embodiment, the LLOprotein is a fragment of an isomer of SEQ ID NO: 37. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the LLO protein utilized to construct vaccines ofmethods and compositions the present invention has the sequence as setforth in SEQ ID NO: 46 (Example 5 hereinbelow). In another embodiment,the LLO protein is a variant of SEQ ID NO: 46. In another embodiment,the LLO protein is an isomer of SEQ ID NO: 46. In another embodiment,the LLO protein is a fragment of SEQ ID NO: 46. In another embodiment,the LLO protein is a fragment of a homologue of SEQ ID NO: 46. Inanother embodiment, the LLO protein is a fragment of a variant of SEQ IDNO: 46. In another embodiment, the LLO protein is a fragment of anisomer of SEQ ID NO: 46.

In another embodiment, the LLO protein utilized to construct vaccines ofmethods and compositions as provided herein is a detoxified LLO (DTLLO).In another embodiment, the DTLLO is a fragment of the full proteinthereof. In another embodiment, LLO is detoxified by replacing thecholesterol binding region with an antigen peptide or epitope thereof.In another embodiment, LLO detoxified by replacing the cholesterolbinding region with the E7 epitope. In another embodiment, LLO isdetoxified by deleting the cholesterol binding region/domain. In anotherembodiment, LLO is detoxified by deleting the signal sequence portion ofLLO. In another embodiment, LLO is detoxified by deleting the signalsequence and the cholesterol binding region/domain. In anotherembodiment, DTLLO is used in genetic or chemical fusions to targetantigens to increase antigen immunogenicity. In another embodiment,detoxLLO is fused to an antigen. In another embodiment, DTLLO is fusedto an antigenic peptide of the methods and compositions describedherein.

In one embodiment, the cholesterol binding region or cholesterol bindingdomain is known as for LLO or may be deduced using methods known in theArt (reviewed in Alouf, Int J Med Microbiol. 2000 October;290(4-5):351-6, incorporated herein by reference), includingsite-directed mutagenesis followed by a cholesterol binding assay orsequence conservation of proteins with similar cholesterol-bindingfunctions.

In another embodiment, the LLO protein is a ctLLO. In another embodimentctLLO is full length LLO in which the CBD has been replaced by anantigen peptide or epitope thereof. In another embodiment “replaced” incan mean via a substitution, or deletion mutation. In anotherembodiment, the LLO protein is a mutLLO. In another embodiment, a mutLLOis one in which the CBD has been mutated. In another embodiment, themutLLO is one in which the amino acids in the CBD have been mutated. Inanother embodiment the mutation is a point mutation, a deletion, aninversion, a substitution, or a combination thereof. In anotherembodiment the mutation is any mutation known in the art. In anotherembodiment, the mutated LLO protein comprises any combination ofdeletions, substitutions, or point mutations in the CBD and/or deletionsof the signal sequence of LLO. In another embodiment, mutating the CBDreduces the hemolytic activity of LLO. In another embodiment, the CBD isreplaced by known HLA class I restricted epitopes to be used as avaccine. In another embodiment, the mutated LLO is expressed andpurified from E. coli expression systems.

In another embodiment, “LLO fragment” or “ΔLLO” refers to a fragment ofLLO that comprises the PEST-like domain thereof. In another embodiment,the terms refer to an LLO fragment that comprises a PEST sequence. Eachpossibility represents another embodiment of the present invention.

In another embodiment, the LLO fragment contains residues of ahomologous LLO protein that correspond to one of the above AA ranges.The residue numbers need not, in another embodiment, correspond exactlywith the residue numbers enumerated above; e.g. if the homologous LLOprotein has an insertion or deletion, relative to an LLO proteinutilized herein.

In another embodiment, the LLO fragment is any other LLO fragment knownin the art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, a whole LLO protein is utilized in methods andcompositions of the present invention. In another embodiment, the wholeLLO protein is a non-hemolytic LLO protein.

In another embodiment, a recombinant peptide, protein or polypeptide ofthe present invention further comprises a detectable tag polypeptide. Inanother embodiment, a detectable tag polypeptide is not included. Inother embodiments, the tag polypeptide is green fluorescent protein(GFP), myc, myc-pyruvate kinase (myc-PK), His₆, maltose biding protein(MBP), an influenza virus hemagglutinin tag polypeptide, a flag tagpolypeptide (FLAG), and a glutathione-S-transferase (GST) tagpolypeptide. However, the invention should in no way be construed to belimited to the nucleic acids encoding the above-listed tag polypeptides.In another embodiment, the present invention utilizes any nucleic acidsequence encoding a polypeptide which functions in a mannersubstantially similar to these tag polypeptides. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the recombinant vaccine vector of methods andcompositions of the present invention is a plasmid. In anotherembodiment, the present invention provides a method for the introductionof a nucleotide molecule of the present invention into a cell. Methodsfor constructing and utilizing recombinant vectors are well known in theart and are described, for example, in Sambrook et al. (2001, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York),and in Brent et al. (2003, Current Protocols in Molecular Biology, JohnWiley & Sons, New York). In another embodiment, the vector is abacterial vector. In other embodiments, the vector is selected fromSalmonella sp., Shigella sp., BCG, L. monocytogenes and S. gordonii. Inanother embodiment, the fusion proteins are delivered by recombinantbacterial vectors modified to escape phagolysosomal fusion and live inthe cytoplasm of the cell. In another embodiment, the vector is a viralvector. In other embodiments, the vector is selected from Vaccinia,Avipox, Adenovirus, AAV, Vaccinia virus NYVAC, Modified vaccinia strainAnkara (MVA), Semliki Forest virus, Venezuelan equine encephalitisvirus, herpes viruses, and retroviruses. In another embodiment, thevector is a naked DNA vector. In another embodiment, the vector is anyother vector known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a nucleotide of the present invention is operablylinked to a promoter/regulatory sequence that drives expression of theencoded peptide in cells into which the vector is introduced.Promoter/regulatory sequences useful for driving constitutive expressionof a gene in a prokaryotic cell are well known in the art and include,for example, the Listeria p60 promoter, the inlA (encodes internalin)promoter, the hly promoter, and the ActA promoter is used. In anotherembodiment, any other gram positive promoter is used.Promoter/regulatory sequences useful for driving constitutive expressionof a gene in a eukaryotic cell (e.g. for a DNA vaccine) are well knownin the art and include, but are not limited to, for example, thecytomegalovirus immediate early promoter enhancer sequence, the SV40early promoter, and the Rous sarcoma virus promoter. In anotherembodiment, inducible and tissue specific expression of the nucleic acidencoding a peptide of the present invention is accomplished by placingthe nucleic acid encoding the peptide under the control of an inducibleor tissue specific promoter/regulatory sequence. Examples of tissuespecific or inducible promoter/regulatory sequences which are useful forhis purpose include, but are not limited to the MMTV LTR induciblepromoter, and the SV40 late enhancer/promoter. In another embodiment, apromoter that is induced in response to inducing agents such as metals,glucocorticoids, and the like, is utilized. Thus, it will be appreciatedthat the invention includes the use of any promoter/regulatory sequence,which is either known or unknown, and which is capable of drivingexpression of the desired protein operably linked thereto.

In another embodiment, a peptide of the present invention activates anAPC (e.g. a DC), mediating at least part of its increasedimmunogenicity. In another embodiment, the inactivated LLO need not beattached to the idiotype-containing protein to enhance itsimmunogenicity. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method forenhancing the immunogenicity of an antigen, comprising fusing an LLOprotein or fragment thereof to the antigen. As demonstrated by the datadisclosed herein, fusing a mutated LLO protein to an antigen enhancesthe immunogenicity of the antigen.

In another embodiment of methods and compositions of the presentinvention, a PEST-like AA sequence is contained in an LLO fusion proteinof the present invention. As provided herein, enhanced cell mediatedimmunity was demonstrated for fusion proteins comprising an antigen andLLO containing the PEST-like AA sequenceKENSISSMAPPASPPASPKTPIEKKHADEIDK (SEQ ID NO: 63). In another embodiment,fusion of an antigen to a non-hemolytic LLO including the PEST-like AAsequence, SEQ ID NO: 1, can enhance cell mediated and anti-tumorimmunity of the antigen.

In another embodiment, the non-hemolytic LLO protein or fragment thereofof the present invention need not be that which is set forth exactly inthe sequences set forth herein, but rather that other alterations,modifications, or changes can be made that retain the functionalcharacteristics of an LLO fused to an antigen as set forth elsewhereherein. In another embodiment, the present invention utilizes an analogof an LLO protein or fragment thereof of the present invention. Analogsdiffer, in another embodiment, from naturally occurring proteins orpeptides by conservative AA sequence differences or by modificationsthat do not affect sequence, or by both.

In one embodiment, the present invention provides a composition ormethod in which cytokine expression is increased (see for e.g., Example19). In one embodiment, the cytokine is TNF-alpha, while in anotherembodiment, the cytokine is IL-12, while in another embodiment, thecytokine is ISG15, while in another embodiment, the cytokine is adifferent cytokine known in the art. In one embodiment, the increase maybe in cytokine mRNA expression, while in another embodiment, it may bein cytokine secretion, while in another embodiment, the increase may bein both mRNA expression and secretion of cytokines. In anotherembodiment, compositions and methods of the present invention mayincrease dendritic cell maturation markers, which in one embodiment, isCD86, in another embodiment, CD40, and in another embodiment MHCII, inanother embodiment, another dendritic cell maturation marker known inthe art, or, in another embodiment, a combination thereof (see for e.g.,Example 20). In another embodiment, compositions and method of thepresent invention may cause nuclear translocation of transcriptionfactors, which in one embodiment, is NF-kappa-B (see for e.g. Example22), or in another embodiment, is a different transcription factor knownin the art. In another embodiment, compositions and method of thepresent invention may cause upregulation of cell surface markers, whichin one embodiment, may be CD11b, which in one embodiment isIntegrin-alpha M (ITGAM); cluster of differentiation molecule 11B;complement receptor 3A (CR3A); or macrophage 1 antigen (MAC-1)A. Inanother embodiment, a different cell surface marker expressed by immunecells, may be upregulated as would be understood by a skilled artisan.

In one embodiment, “homology” refers to identity to an LLO sequence(e.g. to any of SEQ ID NO: 37, 46, or 48) of greater than 70%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:37, 46, or 48 of greater than 72%. In another embodiment, “homology”refers to identity to any of SEQ ID NO: 37, 46, or 48 of greater than75%. In another embodiment, “homology” refers to identity to any of SEQID NO: 37, 46, or 48 of greater than 78%. In another embodiment,“homology” refers to identity to any of SEQ ID NO: 37, 46, or 48 ofgreater than 80%. In another embodiment, “homology” refers to identityto any of SEQ ID NO: 37, 46, or 48 of greater than 82%. In anotherembodiment, “homology” refers to identity to any of SEQ ID NO: 37, 46,or 48 of greater than 83%. In another embodiment, “homology” refers toidentity to any of SEQ ID NO: 37, 46, or 48 of greater than 85%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:37, 46, or 48 of greater than 87%. In another embodiment, “homology”refers to identity to any of SEQ ID NO: 37, 46, or 48 of greater than88%. In another embodiment, “homology” refers to identity to any of SEQID NO: 37, 46, or 48 of greater than 90%. In another embodiment,“homology” refers to identity to any of SEQ ID NO: 37, 46, or 48 ofgreater than 92%. In another embodiment, “homology” refers to identityto any of SEQ ID NO: 37, 46, or 48 of greater than 93%. In anotherembodiment, “homology” refers to identity to any of SEQ ID NO: 37, 46,or 48 of greater than 95%. In another embodiment, “homology” refers toidentity to any of SEQ ID NO: 37, 46, or 48 of greater than 96%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:37, 46, or 48 of greater than 97%. In another embodiment, “homology”refers to identity to any of SEQ ID NO: 37, 46, or 48 of greater than98%. In another embodiment, “homology” refers to identity to any of SEQID NO: 37, 46, or 48 of greater than 99%. In another embodiment,“homology” refers to identity to any of SEQ ID NO: 37, 46, or 48 of100%. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, “homology” refers to identity to acholesterol-binding domain (e.g. to any of SEQ ID NO: 18, 53, or 55) ofgreater than 70%. In another embodiment, “homology” refers to identityto any of SEQ ID NO: 18, 53, or 55 of greater than 72%. In anotherembodiment, “homology” refers to identity to any of SEQ ID NO: 18, 53,or 55 of greater than 75%. In another embodiment, “homology” refers toidentity to any of SEQ ID NO: 18, 53, or 55 of greater than 78%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:18, 53, or 55 of greater than 80%. In another embodiment, “homology”refers to identity to any of SEQ ID NO: 18, 53, or 55 of greater than82%. In another embodiment, “homology” refers to identity to any of SEQID NO: 18, 53, or 55 of greater than 83%. In another embodiment,“homology” refers to identity to any of SEQ ID NO: 18, 53, or 55 ofgreater than 85%. In another embodiment, “homology” refers to identityto any of SEQ ID NO: 18, 53, or 55 of greater than 87%. In anotherembodiment, “homology” refers to identity to any of SEQ ID NO: 18, 53,or 55 of greater than 88%. In another embodiment, “homology” refers toidentity to any of SEQ ID NO: 18, 53, or 55 of greater than 90%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:18, 53, or 55 of greater than 92%. In another embodiment, “homology”refers to identity to any of SEQ ID NO: 18, 53, or 55 of greater than93%. In another embodiment, “homology” refers to identity to any of SEQID NO: 18, 53, or 55 of greater than 95%. In another embodiment,“homology” refers to identity to any of SEQ ID NO: 18, 53, or 55 ofgreater than 96%. In another embodiment, “homology” refers to identityto any of SEQ ID NO: 18, 53, or 55 of greater than 97%. In anotherembodiment, “homology” refers to identity to any of SEQ ID NO: 18, 53,or 55 of greater than 98%. In another embodiment, “homology” refers toidentity to any of SEQ ID NO: 18, 53, or 55 of greater than 99%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:18, 53, or 55 of 100%. Each possibility represents a separate embodimentof the present invention.

In another embodiment, “homology” refers to identity to an NY-ESO-1sequence (e.g. to any of SEQ ID NO: 1-15) of greater than 70%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:1-15 of greater than 72%. In another embodiment, “homology” refers toidentity to any of SEQ ID NO: 1-15 of greater than 75%. In anotherembodiment, “homology” refers to identity to any of SEQ ID NO: 1-15 ofgreater than 78%. In another embodiment, “homology” refers to identityto any of SEQ ID NO: 1-15 of greater than 80%. In another embodiment,“homology” refers to identity to any of SEQ ID NO: 1-15 of greater than82%. In another embodiment, “homology” refers to identity to any of SEQID NO: 1-15 of greater than 83%. In another embodiment, “homology”refers to identity to any of SEQ ID NO: 1-15 of greater than 85%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:1-15 of greater than 87%. In another embodiment, “homology” refers toidentity to any of SEQ ID NO: 1-15 of greater than 88%. In anotherembodiment, “homology” refers to identity to any of SEQ ID NO: 1-15 ofgreater than 90%. In another embodiment, “homology” refers to identityto any of SEQ ID NO: 1-15 of greater than 92%. In another embodiment,“homology” refers to identity to any of SEQ ID NO: 1-15 of greater than93%. In another embodiment, “homology” refers to identity to any of SEQID NO: 1-15 of greater than 95%. In another embodiment, “homology”refers to identity to any of SEQ ID NO: 1-15 of greater than 96%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:1-15 of greater than 97%. In another embodiment, “homology” refers toidentity to any of SEQ ID NO: 1-15 of greater than 98%. In anotherembodiment, “homology” refers to identity to any of SEQ ID NO: 1-15 ofgreater than 99%. In another embodiment, “homology” refers to identityto any of SEQ ID NO: 1-15 of 100%. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, “homology” refers to identity to an E7 sequence(e.g. to any of any of SEQ ID NO: 17, 19-23) of greater than 70%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:17, 19-23 of greater than 72%. In another embodiment, “homology” refersto identity to any of SEQ ID NO: 17, 19-23 of greater than 75%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:17, 19-23 of greater than 78%. In another embodiment, “homology” refersto identity to any of SEQ ID NO: 17, 19-23 of greater than 80%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:17, 19-23 of greater than 82%. In another embodiment, “homology” refersto identity to any of SEQ ID NO: 17, 19-23 of greater than 83%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:17, 19-23 of greater than 85%. In another embodiment, “homology” refersto identity to any of SEQ ID NO: 17, 19-23 of greater than 87%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:17, 19-23 of greater than 88%. In another embodiment, “homology” refersto identity to any of SEQ ID NO: 17, 19-23 of greater than 90%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:17, 19-23 of greater than 92%. In another embodiment, “homology” refersto identity to any of SEQ ID NO: 17, 19-23 of greater than 93%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:17, 19-23 of greater than 95%. In another embodiment, “homology” refersto identity to any of SEQ ID NO: 17, 19-23 of greater than 96%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:17, 19-23 of greater than 97%. In another embodiment, “homology” refersto identity to any of SEQ ID NO: 17, 19-23 of greater than 98%. Inanother embodiment, “homology” refers to identity to any of SEQ ID NO:17, 19-23 of greater than 99%. In another embodiment, “homology” refersto identity to any of SEQ ID NO: 17, 19-23 of 100%. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, “homology” refers to identity to a BCR sequence(e.g. to any of any one of SEQ ID NO: 26-36) of greater than 70%. Inanother embodiment, “homology” refers to identity to any one of SEQ IDNO: 26-36 of greater than 72%. In another embodiment, “homology” refersto identity to any one of SEQ ID NO: 26-36 of greater than 75%. Inanother embodiment, “homology” refers to identity to any one of SEQ IDNO: 26-36 of greater than 78%. In another embodiment, “homology” refersto identity to any one of SEQ ID NO: 26-36 of greater than 80%. Inanother embodiment, “homology” refers to identity to any one of SEQ IDNO: 26-36 of greater than 82%. In another embodiment, “homology” refersto identity to any one of SEQ ID NO: 26-36 of greater than 83%. Inanother embodiment, “homology” refers to identity to any one of SEQ IDNO: 26-36 of greater than 85%. In another embodiment, “homology” refersto identity to any one of SEQ ID NO: 26-36 of greater than 87%. Inanother embodiment, “homology” refers to identity to any one of SEQ IDNO: 26-36 of greater than 88%. In another embodiment, “homology” refersto identity to any one of SEQ ID NO: 26-36 of greater than 90%. Inanother embodiment, “homology” refers to identity to any one of SEQ IDNO: 26-36 of greater than 92%. In another embodiment, “homology” refersto identity to any one of SEQ ID NO: 26-36 of greater than 93%. Inanother embodiment, “homology” refers to identity to any one of SEQ IDNO: 26-36 of greater than 95%. In another embodiment, “homology” refersto identity to any one of SEQ ID NO: 26-36 of greater than 96%. Inanother embodiment, “homology” refers to identity to any one of SEQ IDNO: 26-36 of greater than 97%. In another embodiment, “homology” refersto identity to any one of SEQ ID NO: 26-36 of greater than 98%. Inanother embodiment, “homology” refers to identity to any one of SEQ IDNO: 26-36 of greater than 99%. In another embodiment, “homology” refersto identity to any one of SEQ ID NO: 26-36 of 100%. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment of the present invention, “nucleic acids” or“nucleotide” refers to a string of at least two base-sugar-phosphatecombinations. The term includes, in one embodiment, DNA and RNA.“Nucleotides” refers, in one embodiment, to the monomeric units ofnucleic acid polymers. RNA is, in one embodiment, in the form of a tRNA(transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA(messenger RNA), anti-sense RNA, small inhibitory RNA (siRNA), micro RNA(miRNA) and ribozymes. The use of siRNA and miRNA has been described(Caudy A A et al, Genes & Devel 16: 2491-96 and references citedtherein). In other embodiments, DNA can be in form of plasmid DNA, viralDNA, linear DNA, or chromosomal DNA or derivatives of these groups. Inaddition, these forms of DNA and RNA can be single, double, triple, orquadruple stranded. The term also includes, in another embodiment,artificial nucleic acids that contain other types of backbones but thesame bases. In one embodiment, the artificial nucleic acid is a PNA(peptide nucleic acid). PNA contain peptide backbones and nucleotidebases and are able to bind, in one embodiment, to both DNA and RNAmolecules. In another embodiment, the nucleotide is oxetane modified. Inanother embodiment, the nucleotide is modified by replacement of one ormore phosphodiester bonds with a phosphorothioate bond. In anotherembodiment, the artificial nucleic acid contains any other variant ofthe phosphate backbone of native nucleic acids known in the art. The useof phosphothiorate nucleic acids and PNA are known to those skilled inthe art, and are described in, for example, Neilsen P E, Curr OpinStruct Biol 9:353-57; and Raz N K et al Biochem Biophys Res Commun297:1075-84. The production and use of nucleic acids is known to thoseskilled in art and is described, for example, in Molecular Cloning,(2001), Sambrook and Russell, eds. and Methods in Enzymology: Methodsfor molecular cloning in eukaryotic cells (2003) Purchio and G. C.Fareed. Each nucleic acid derivative represents a separate embodiment ofthe present invention.

Protein and/or peptide homology for any AA sequence listed herein isdetermined, in one embodiment, by methods well described in the art,including immunoblot analysis, or via computer algorithm analysis of AAsequences, utilizing any of a number of software packages available, viaestablished methods. Some of these packages include the FASTA, BLAST,MPsrch or Scanps packages, and employ, in other embodiments, the use ofthe Smith and Waterman algorithms, and/or global/local or BLOCKSalignments for analysis, for example. Each method of determininghomology represents a separate embodiment of the present invention.

In another embodiment, a recombinant peptide, protein or polypeptide ofthe present invention is made by a process that comprises the step ofchemically conjugating a peptide comprising the LLO protein or fragmentthereof to a peptide comprising the antigen. In another embodiment, anLLO protein or fragment thereof is chemically conjugated to a peptidecomprising the antigen. In another embodiment, a peptide comprising theLLO protein or fragment thereof is chemically conjugated to the antigen.In another embodiment, the LLO protein or fragment thereof is chemicallyconjugated to the antigen. Each possibility represents a separateembodiment of the present invention.

“Peptide” refers, in another embodiment, to a chain of AA connected withpeptide bonds. In one embodiment, a peptide is a short chain of AAs. Inanother embodiment, the term refers to a variant peptide molecule,containing any modification disclosed or enumerated herein. In anotherembodiment, the term refers to a molecule containing one or moremoieties introduced by a chemical cross-linker. In another embodiment,the term refers to a peptide mimetic molecule. In another embodiment,the term refers to any other type of variant of a peptide molecule knownin the art. Each possibility represents a separate embodiment of thepresent invention.

In one embodiment, the term “protein” or “polypeptide” is an amino acidchain comprising multiple peptide subunits, including a full-lengthprotein, oligopeptides, and fragments thereof, wherein the amino acidresidues are linked by covalent peptide bonds. In one embodiment, aprotein described in the present invention may alternatively be apolypeptide of the present invention.

As used herein in the specification and in the examples section whichfollows the term “peptide” includes native peptides (either degradationproducts, synthetically synthesized peptides or recombinant peptides)and peptidomimetics (typically, synthetically synthesized peptides),such as peptoids and semipeptoids which are peptide analogs, which mayhave, for example, modifications rendering the peptides more stablewhile in a body or more capable of penetrating into bacterial cells.Such modifications include, but are not limited to N terminusmodification, C terminus modification, peptide bond modification,including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O,CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified, for example, in Quantitative DrugDesign, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press(1992), which is incorporated by reference as if fully set forth herein.Further details in this respect are provided hereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)-CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as TIC, naphthylelanine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe oro-methyl-Tyr.

In addition to the above, the peptides of the present invention may alsoinclude one or more modified amino acids or one or more non-amino acidmonomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification and in the claims section below theterm “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Tables 1 and 2 below list naturally occurring amino acids (Table 1) andnon-conventional or modified amino acids (Table 2) which can be usedwith the present invention.

TABLE 1 Three-Letter Amino Acid Abbreviation One-letter Symbol AlanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic Acid Glu E Glycine Gly G Histidine His HIsoleucine Iie I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid as Xaa Xabove

TABLE 2 Non-conventional Non-conventional amino amino acid Code acidCode α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α- MgabuL-N-methylarginine Nmarg methylbutyrate aminocyclopropane- CproL-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmaspaminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- NorbL-N-methylglutamine Nmgin carboxylate L-N-methylglutamic acid Nmglucyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanineCpen L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine NmleuD-arginine Darg L-N-methyllysine Nmlys D-aspartic acid DaspL-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine NmnleD-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid DgluL-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine NmpheD-isoleucine Dile L-N-methylproline Nmpro D-leucine DleuL-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine NmthrD-methionine Dmet L-N-methyltryptophan Nmtrp D-ornithine DornL-N-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline NmvalD-proline Dpro L-N-methylethylglycine Nmetg D-serine DserL-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine NleD-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcyclopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α- Dmphe N-(2-carboxyethyl)glycine Nglumethylphenylalanine D-α-methylproline Dmpro N-(carboxymethyl)glycineNasp D-α-methylserine Dmser N-cyclobutylglycine NcbutD-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cyclododeclglycine Ncdod D-α-methylalnine Dnmala N-cyclooctylglycineNcoct D-α-methylarginine Dnmarg N-cyclopropylglycine NcproD-α-methylasparagine Dnmasn N-cycloundecylglycine NcundD-α-methylasparatate Dnmasp N-(2,2- Nbhm diphenylethyl)glycineD-α-methylcysteine Dnmcys N-(3,3- Nbhe diphenylpropyl)glycineD-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N- NmchexaD-N-methylmethionine Dnmmet methylcyclohexylalanine D-N-methylornithineDnmorn N- Nmcpen methylcyclopentylalanine N-methylglycine NalaD-N-methylphenylalanine Dnmphe N- Nmaib D-N-methylproline Dnmpromethylaminoisobutyrate N-(1- Nile D-N-methylserine Dnmsermethylpropyl)glycine N-(2- Nile D-N-methylserine Dnmsermethylpropyl)glycine N-(2- Nleu D-N-methylthreonine Dnmthrmethylpropyl)glycine D-N-methyltryptophan DnmtrpN-(1-methylethyl)glycine Nva D-N-methyltyrosine DnmtyrN-methyla-napthylalanine Nmanap D-N-methylvaline DnmvalN-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p- Nhtyrhydroxyphenyl)glycine L-t-butylglycine Tbug N-(thiomethyl)glycine NcysL-ethylglycine Etg penicillamine Pen L-homophenylalanine HpheL-α-methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine MasnL-α-methylaspartate Masp L-α-methyl-t-butylglycine MtbugL-α-methylcysteine Mcys L-methylethylglycine Metg L-α-methylglutamineMgln L-α-methylglutamate Mglu L-α-methylhistidine Mhis L-α-methylhomoMhphe phenylalanine L-α-methylisoleucine Mile N-(2- Nmetmethylthioethyl)glycine D-N-methylglutamine Dnmgln N-(3- Nargguanidinopropyl)glycine D-N-methylglutamate DnmgluN-(1-hydroxyethyl)glycine Nthr D-N-methylhistidine DnmhisN-(hydroxyethyl)glycine Nser D-N-methylisoleucine DnmileN-(imidazolylethyl)glycine Nhis D-N-methylleucine DnmleuN-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine DnmlysN-methyl-γ-aminobutyrate Nmgabu N- Nmchexa D-N-methylmethionine Dnmmetmethylcyclohexylalanine D-N-methylornithine Dnmorn N- Nmcpenmethylcyclopentylalanine N-methylglycine Nala D-N-methylphenylalanineDnmphe N- Nmaib D-N-methylproline Dnmpro methylaminoisobutyrate N-(1-Nile D-N-methylserine Dnmser methylpropyl)glycine N-(2- NleuD-N-methylthreonine Dnmthr methylpropyl)glycine D-N-methyltryptophanDnmtrp N-(1-methylethyl)glycine Nval D-N-methyltyrosine DnmtyrN-methyla-napthylalanine Nmanap D-N-methylvaline DnmvalN-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p- Nhtyrhydroxyphenyl)glycine L-t-butylglycine Tbug N-(thiomethyl)glycine NcysL-ethylglycine Etg penicillamine Pen L-homophenylalanine HpheL-α-methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine MasnL-α-methylaspartate Masp L-α-methyl-t-butylglycine MtbugL-α-methylcysteine Mcys L-methylethylglycine Metg L-α-methylglutamineMgln L-α-methylglutamate Mglu L-α-methylhistidine Mhis L-α- Mhphemethylhomophenylalanine L-α-methylisoleucine Mile N-(2- Nmetmethylthioethyl)glycine L-α-methylleucine Mleu L-α-methyllysine MlysL-α-methylmethionine Mmet L-α-methylnorleucine Mnle L-α-methylnorvalineMnva L-α-methylornithine Morn L-α-methylphenylalanine MpheL-α-methylproline Mpro L-α-methylserine mser L-α-methylthreonine MthrL-α-methylvaline Mtrp L-α-methyltyrosine Mtyr L-α-methylleucine MvalNnbhm L-N- Nmhphe methylhomophenylalanineN-(N-(2,2-diphenylethyl)carbamylmethyl- NnbhmN-(N-(3,3-diphenylpropyl)carbamylmethyl(1)glycine Nnbhe glycine1-carboxy-1-(2,2- Nmbc diphenyl ethylamino)cyclopropane

In another embodiment, the method used for conjugating the non-hemolyticLLO protein or fragment thereof to the antigen is that described inExample 11. In another embodiment, another method known in the art isutilized. Methods for chemical conjugation of peptides to one anotherare well known in the art, and are described for, example, in (Biragyn,A and Kwak, L W (2001) Mouse models for lymphoma in “Current Protocolsin Immunology” 20.6.1-20.6.30) and (Collawn, J. F. and Paterson, Y.(1989) Preparation of Anti-peptide antibodies. In Current Protocols inMolecular Biology. Supplement 6. Ed. F. M. Ausubel et. al. GreenePublishing/Wiley 11.14.1-11.15.3).

In another embodiment, the non-hemolytic LLO protein or fragment thereofor N-terminal LLO fragment is attached to the antigen or fragmentthereof by chemical conjugation. In another embodiment, thenon-hemolytic LLO protein or fragment thereof or N-terminal LLO fragmentis attached to the heterologous peptide by chemical conjugation. Inanother embodiment, glutaraldehyde is used for the conjugation. Inanother embodiment, the conjugation is performed using any suitablemethod known in the art. Each possibility represents another embodimentof the present invention.

In another embodiment, a fusion peptide of the present invention issynthesized using standard chemical peptide synthesis techniques. Inanother embodiment, the chimeric molecule is synthesized as a singlecontiguous polypeptide. In another embodiment, the LLO protein, ActAprotein, or fragment thereof; and the BCR or fragment thereof aresynthesized separately, then fused by condensation of the amino terminusof one molecule with the carboxyl terminus of the other molecule,thereby forming a peptide bond. In another embodiment, the ActA proteinor LLO protein and antigen are each condensed with one end of a peptidespacer molecule, thereby forming a contiguous fusion protein.

In another embodiment, fusion proteins of the present invention areprepared by any suitable method, including, for example, cloning andrestriction of appropriate sequences or direct chemical synthesis bymethods discussed below. In another embodiment, subsequences are clonedand the appropriate subsequences cleaved using appropriate restrictionenzymes. The fragments are then ligated, in another embodiment, toproduce the desired DNA sequence. In another embodiment, DNA encodingthe fusion protein is produced using DNA amplification methods, forexample polymerase chain reaction (PCR). First, the segments of thenative DNA on either side of the new terminus are amplified separately.The 5′ end of the one amplified sequence encodes the peptide linker,while the 3′ end of the other amplified sequence also encodes thepeptide linker. Since the 5′ end of the first fragment is complementaryto the 3′ end of the second fragment, the two fragments (after partialpurification, e.g. on LMP agarose) can be used as an overlappingtemplate in a third PCR reaction. The amplified sequence will containcodons, the segment on the carboxy side of the opening site (now formingthe amino sequence), the linker, and the sequence on the amino side ofthe opening site (now forming the carboxyl sequence). The insert is thenligated into a plasmid.

In another embodiment, a recombinant peptide, protein or polypeptide ofthe present invention is synthesized using standard chemical peptidesynthesis techniques. In another embodiment, the chimeric molecule issynthesized as a single contiguous polypeptide. In another embodiment,the non-hemolytic LLO protein or fragment thereof; and the antigen aresynthesized separately, then fused by condensation of the amino terminusof one molecule with the carboxyl terminus of the other molecule,thereby forming a peptide bond. In another embodiment, the LLO proteinand antigen are each condensed with one end of a peptide spacermolecule, thereby forming a contiguous fusion protein.

In another embodiment, the peptides and proteins of the presentinvention are prepared by solid-phase peptide synthesis (SPPS) asdescribed by Stewart et al. in Solid Phase Peptide Synthesis, 2ndEdition, 1984, Pierce Chemical Company, Rockford, Ill.; or as describedby Bodanszky and Bodanszky (The Practice of Peptide Synthesis, 1984,Springer-Verlag, New York). In another embodiment, a suitably protectedAA residue is attached through its carboxyl group to a derivatized,insoluble polymeric support, such as cross-linked polystyrene orpolyamide resin. “Suitably protected” refers to the presence ofprotecting groups on both the alpha-amino group of the amino acid, andon any side chain functional groups. Side chain protecting groups aregenerally stable to the solvents, reagents and reaction conditions usedthroughout the synthesis, and are removable under conditions which willnot affect the final peptide product. Stepwise synthesis of theoligopeptide is carried out by the removal of the N-protecting groupfrom the initial AA, and couple thereto of the carboxyl end of the nextAA in the sequence of the desired peptide. This AA is also suitablyprotected. The carboxyl of the incoming AA can be activated to reactwith the N-terminus of the support-bound AA by formation into a reactivegroup such as formation into a carbodiimide, a symmetric acid anhydrideor an “active ester” group such as hydroxybenzotriazole orpentafluorophenly esters.

Examples of solid phase peptide synthesis methods include the BOC methodwhich utilized tert-butyloxcarbonyl as the alpha-amino protecting group,and the FMOC method which utilizes 9-fluorenylmethyloxcarbonyl toprotect the alpha-amino of the AA residues, both methods of which arewell-known by those of skill in the art.

In another embodiment, incorporation of N- and/or C-blocking groups isachieved using protocols conventional to solid phase peptide synthesismethods. For incorporation of C-terminal blocking groups, for example,synthesis of the desired peptide is typically performed using, as solidphase, a supporting resin that has been chemically modified so thatcleavage from the resin results in a peptide having the desiredC-terminal blocking group. To provide peptides in which the C-terminusbears a primary amino blocking group, for instance, synthesis isperformed using a p-methylbenzhydrylamine (MBHA) resin so that, whenpeptide synthesis is completed, treatment with hydrofluoric acidreleases the desired C-terminally amidated peptide. Similarly,incorporation of an N-methylamine blocking group at the C-terminus isachieved using N-methylaminoethyl-derivatized DVB, resin, which upon HFtreatment releases a peptide bearing an N-methylamidated C-terminusBlockage of the C-terminus by esterification can also be achieved usingconventional procedures. This entails use of resin/blocking groupcombination that permits release of side-chain peptide from the resin,to allow for subsequent reaction with the desired alcohol, to form theester function. FMOC protecting group, in combination with DVB resinderivatized with methoxyalkoxybenzyl alcohol or equivalent linker, canbe used for this purpose, with cleavage from the support being effectedby TFA in dicholoromethane. Esterification of the suitably activatedcarboxyl function e.g. with DCC, can then proceed by addition of thedesired alcohol, followed by deprotection and isolation of theesterified peptide product.

Incorporation of N-terminal blocking groups can be achieved while thesynthesized peptide is still attached to the resin, for instance bytreatment with a suitable anhydride and nitrile. To incorporate anacetyl blocking group at the N-terminus, for instance, the resin coupledpeptide can be treated with 20% acetic anhydride in acetonitrile. TheN-blocked peptide product can then be cleaved from the resin,deprotected and subsequently isolated.

In another embodiment, analysis of the peptide composition is conductedto verify the identity of the produced peptide. In another embodiment,AA composition analysis is conducted using high-resolution massspectrometry to determine the molecular weight of the peptide.Alternatively, or additionally, the AA content of the peptide isconfirmed by hydrolyzing the peptide in aqueous acid, and separating,identifying and quantifying the components of the mixture using HPLC, oran AA analyzer. Protein sequencers, which sequentially degrade thepeptide and identify the AA in order, can also be used to determinedefinitely the sequence of the peptide.

In another embodiment, prior to its use, the peptide is purified toremove contaminants. In another embodiment, the peptide is purified soas to meet the standards set out by the appropriate regulatory agenciesand guidelines. Any one of a number of a conventional purificationprocedures can be used to attain the required level of purity,including, for example, reversed-phase high-pressure liquidchromatography (HPLC) using an alkylated silica column such as C₄-, C₈-or C₁₈-silica. A gradient mobile phase of increasing organic content isgenerally used to achieve purification, for example, acetonitrile in anaqueous buffer, usually containing a small amount of trifluoroaceticacid. Ion-exchange chromatography can be also used to separate peptidesbased on their charge.

Solid phase synthesis in which the C-terminal AA of the sequence isattached to an insoluble support followed by sequential addition of theremaining AA in the sequence is used, in another embodiment, for thechemical synthesis of the peptides of this invention. Techniques forsolid phase synthesis are described by Barany and Merrifield inSolid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), andStewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill. (1984).

In another embodiment, fusion proteins of the present invention aresynthesized using recombinant DNA methodology. In another embodiment,DNA encoding the fusion protein of the present invention is prepared byany suitable method, including, for example, cloning and restriction ofappropriate sequences or direct chemical synthesis by methods such asthe phosphotriester method of Narang et al. (1979, Meth. Enzymol. 68:90-99); the phosphodiester method of Brown et al. (1979, Meth. Enzymol68: 109-151); the diethylphosphoramidite method of Beaucage et al.(1981, Tetra. Lett., 22: 1859-1862); and the solid support method ofU.S. Pat. No. 4,458,066.

In another embodiment, peptides of the present invention incorporate AAresidues that are modified without affecting activity. In anotherembodiment, the termini are derivatized to include blocking groups, i.e.chemical substituents suitable to protect and/or stabilize the N- andC-termini from “undesirable degradation”, a term meant to encompass anytype of enzymatic, chemical or biochemical breakdown of the compound atits termini which is likely to affect the function of the compound, i.e.sequential degradation of the compound at a terminal end thereof.

In another embodiment, blocking groups include protecting groupsconventionally used in the art of peptide chemistry that will notadversely affect the in vivo activities of the peptide. For example,suitable N-terminal blocking groups can be introduced by alkylation oracylation of the N-terminus Examples of suitable N-terminal blockinggroups include C₁-C₅ branched or unbranched alkyl groups, acyl groupssuch as formyl and acetyl groups, as well as substituted forms thereof,such as the acetamidomethyl (Acm) group. Desamino AA analogs are alsouseful N-terminal blocking groups, and can either be coupled to theN-terminus of the peptide or used in place of the N-terminal reside.Suitable C-terminal blocking groups, in which the carboxyl group of theC-terminus is either incorporated or not, include esters, ketones oramides. Ester or ketone-forming alkyl groups, particularly lower alkylgroups such as methyl, ethyl and propyl, and amide-forming amino groupssuch as primary amines (—NH₂), and mono- and di-alkyl amino groups suchas methyl amino, ethylamino, dimethylamino, diethylamino,methylethylamino and the like are examples of C-terminal blockinggroups. Descarboxylated AA analogues such as agmatine are also usefulC-terminal blocking groups and can be either coupled to the peptide'sC-terminal residue or used in place of it. In another embodiment, thefree amino and carboxyl groups at the termini are removed altogetherfrom the peptide to yield desamino and descarboxylated forms thereofwithout affect on peptide activity.

In another embodiment, other modifications are incorporated withoutadversely affecting the activity. In another embodiment, suchmodifications include, but are not limited to, substitution of one ormore of the AA in the natural L-isomeric form with D-isomeric AA. Inanother embodiment, the peptide includes one or more D-amino acidresides, or comprises AA that are all in the D-form. Retro-inverso formsof peptides in accordance with the present invention are alsocontemplated, for example, inverted peptides in which all amino acidsare substituted with D-amino acid forms.

In another embodiment, acid addition salts peptides of the presentinvention are utilized as functional equivalents thereof. In anotherembodiment, a peptide in accordance with the present invention treatedwith an inorganic acid such as hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, and the like, or an organic acid such as an acetic,propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic,fumaric, tataric, citric, benzoic, cinnamie, mandelic, methanesulfonic,ethanesulfonic, p-toluenesulfonic, salicyclic and the like, to provide awater soluble salt of the peptide is suitable for use in the invention.

In another embodiment, modifications (which do not normally alterprimary sequence) include in vivo, or in vitro chemical derivatizationof polypeptides, e.g., acetylation, or carboxylation. Also included aremodifications of glycosylation, e.g., those made by modifying theglycosylation patterns of a polypeptide during its synthesis andprocessing or in further processing steps; e.g., by exposing thepolypeptide to enzymes which affect glycosylation, e.g., mammalianglycosylating or deglycosylating enzymes. Also embraced are sequenceswhich have phosphorylated AA residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

In another embodiment, polypeptides are modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. Analogs of suchpolypeptides include those containing residues other than naturallyoccurring L-amino acids, e.g., D-amino acids or non-naturally occurringsynthetic amino acids. The peptides of the invention are not limited toproducts of any of the specific exemplary processes listed herein.

In another embodiment, the present invention provides a kit comprisingan non-hemolytic LLO protein or fragment thereof fused to an antigen, anapplicator, and instructional material that describes use of the methodsof the invention. Although model kits are described below, the contentsof other useful kits will be apparent to the skilled artisan in light ofthe present disclosure. Each of these kits is contemplated within thepresent invention.

In another embodiment, the Listeria strain of methods and compositionsof the present invention is a recombinant Listeria seeligeri strain. Inanother embodiment, the Listeria strain is a recombinant Listeria grayistrain. In another embodiment, the Listeria strain is a recombinantListeria ivanovii strain. In another embodiment, the Listeria strain isa recombinant Listeria murrayi strain. In another embodiment, theListeria strain is a recombinant Listeria welshimeri strain. In anotherembodiment, the Listeria strain is a recombinant strain of any otherListeria species known in the art. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a recombinant Listeria strain of the presentinvention has been passaged through an animal host. In anotherembodiment, the passaging maximizes efficacy of the strain as a vaccinevector. In another embodiment, the passaging stabilizes theimmunogenicity of the Listeria strain. In another embodiment, thepassaging stabilizes the virulence of the Listeria strain. In anotherembodiment, the passaging increases the immunogenicity of the Listeriastrain. In another embodiment, the passaging increases the virulence ofthe Listeria strain. In another embodiment, the passaging removesunstable sub-strains of the Listeria strain. In another embodiment, thepassaging reduces the prevalence of unstable sub-strains of the Listeriastrain. In another embodiment, the Listeria strain contains a genomicinsertion of the gene encoding a recombinant peptide, protein orpolypeptide of the present invention. In another embodiment, theListeria strain carries a plasmid comprising the gene encoding arecombinant peptide, protein or polypeptide of the present invention.Methods for passaging a recombinant Listeria strain through an animalhost are well known in the art, and are described, for example, inUnited States Patent Application No. 2006/0233835, which is incorporatedherein by reference. In another embodiment, the passaging is performedby any other method known in the art. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the recombinant Listeria strain utilized inmethods of the present invention has been stored in a frozen cell bank.In another embodiment, the recombinant Listeria strain has been storedin a lyophilized cell bank. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the cell bank of methods and compositions of thepresent invention is a master cell bank. In another embodiment, the cellbank is a working cell bank. In another embodiment, the cell bank isGood Manufacturing Practice (GMP) cell bank. In another embodiment, thecell bank is intended for production of clinical-grade material. Inanother embodiment, the cell bank conforms to regulatory practices forhuman use. In another embodiment, the cell bank is any other type ofcell bank known in the art. Each possibility represents a separateembodiment of the present invention.

“Good Manufacturing Practices” are defined, in another embodiment, by(21 CFR 210-211) of the United States Code of Federal Regulations. Inanother embodiment, “Good Manufacturing Practices” are defined by otherstandards for production of clinical-grade material or for humanconsumption; e.g. standards of a country other than the United States.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a recombinant Listeria strain utilized in methodsof the present invention is from a batch of vaccine doses.

In another embodiment, a recombinant Listeria strain utilized in methodsof the present invention is from a frozen stock produced by a methoddisclosed herein.

In another embodiment, a recombinant Listeria strain utilized in methodsof the present invention is from a lyophilized stock produced by amethod disclosed herein. Methods for lyophilizing recombinant Listeriastrains are well known in the art, and are described, for example, inPCT International Patent Application Publication No. WO 2007/061848.Each method represents a separate embodiment of the present invention.

In another embodiment, a cell bank, frozen stock, or batch of vaccinedoses of the present invention is cryopreserved by a method thatcomprises growing a culture of the Listeria strain in a nutrient media,freezing the culture in a solution comprising glycerol, and storing theListeria strain at below −20 degrees Celsius. In another embodiment, thetemperature is about −70 degrees Celsius. In another embodiment, thetemperature is about ⁻70-⁻80 degrees Celsius.

In another embodiment, a cell bank, frozen stock, or batch of vaccinedoses of the present invention is cryopreserved by a method thatcomprises growing a culture of the Listeria strain in a defined media ofthe present invention (as described below), freezing the culture in asolution comprising glycerol, and storing the Listeria strain at below−20 degrees Celsius. In another embodiment, the temperature is about −70degrees Celsius. In another embodiment, the temperature is about ⁻70-⁻80degrees Celsius. Methods for cryopreservation of recombinant Listeriastrains are well known in the art, and are described, for example, inPCT International Patent Application Publication No. WO 2007/061848.Each method represents a separate embodiment of the present invention.

In another embodiment, any defined microbiological media of the presentinvention may be used in this method. Each defined microbiological mediarepresents a separate embodiment of the present invention.

In another embodiment of methods and compositions of the presentinvention, the culture (e.g. the culture of a Listeria vaccine strainthat is used to produce a batch of Listeria vaccine doses) is inoculatedfrom a cell bank. In another embodiment, the culture is inoculated froma frozen stock. In another embodiment, the culture is inoculated from astarter culture. In another embodiment, the culture is inoculated from acolony. In another embodiment, the culture is inoculated at mid-loggrowth phase. In another embodiment, the culture is inoculated atapproximately mid-log growth phase. In another embodiment, the cultureis inoculated at another growth phase. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the solution used for freezing contains anothercolligative additive or additive with anti-freeze properties, in placeof glycerol. In another embodiment, the solution used for freezingcontains another colligative additive or additive with anti-freezeproperties, in addition to glycerol. In another embodiment, the additiveis mannitol. In another embodiment, the additive is DMSO. In anotherembodiment, the additive is sucrose. In another embodiment, the additiveis any other colligative additive or additive with anti-freezeproperties that is known in the art. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the nutrient media utilized for growing a cultureof a Listeria strain is LB. In another embodiment, the nutrient media isTB. In another embodiment, the nutrient media is a modified,animal-product free Terrific Broth. In another embodiment, the nutrientmedia is a defined media. In another embodiment, the nutrient media is adefined media of the present invention. In another embodiment, thenutrient media is any other type of nutrient media known in the art.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment of methods and compositions of the presentinvention, the step of growing is performed with a shake flask. Inanother embodiment, the flask is a baffled shake flask. In anotherembodiment, the growing is performed with a batch fermenter. In anotherembodiment, the growing is performed with a stirred tank or flask. Inanother embodiment, the growing is performed with an airflit fermenter.In another embodiment, the growing is performed with a fed batch. Inanother embodiment, the growing is performed with a continuous cellreactor. In another embodiment, the growing is performed with animmobilized cell reactor. In another embodiment, the growing isperformed with any other means of growing bacteria that is known in theart. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a constant pH is maintained during growth of theculture (e.g. in a batch fermenter). In another embodiment, the pH ismaintained at about 7.0. In another embodiment, the pH is about 6. Inanother embodiment, the pH is about 6.5. In another embodiment, the pHis about 7.5. In another embodiment, the pH is about 8. In anotherembodiment, the pH is 6.5-7.5. In another embodiment, the pH is 6-8. Inanother embodiment, the pH is 6-7. In another embodiment, the pH is 7-8.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a constant temperature is maintained duringgrowth of the culture. In another embodiment, the temperature ismaintained at about 37° C. In another embodiment, the temperature is 37°C. In another embodiment, the temperature is 25° C. In anotherembodiment, the temperature is 27° C. In another embodiment, thetemperature is 28° C. In another embodiment, the temperature is 30° C.In another embodiment, the temperature is 32° C. In another embodiment,the temperature is 34° C. In another embodiment, the temperature is 35°C. In another embodiment, the temperature is 36° C. In anotherembodiment, the temperature is 38° C. In another embodiment, thetemperature is 39° C. Each possibility represents a separate embodimentof the present invention.

In another embodiment, a constant dissolved oxygen concentration ismaintained during growth of the culture. In another embodiment, thedissolved oxygen concentration is maintained at 20% of saturation. Inanother embodiment, the concentration is 15% of saturation. In anotherembodiment, the concentration is 16% of saturation. In anotherembodiment, the concentration is 18% of saturation. In anotherembodiment, the concentration is 22% of saturation. In anotherembodiment, the concentration is 25% of saturation. In anotherembodiment, the concentration is 30% of saturation. In anotherembodiment, the concentration is 35% of saturation. In anotherembodiment, the concentration is 40% of saturation. In anotherembodiment, the concentration is 45% of saturation. In anotherembodiment, the concentration is 50% of saturation. In anotherembodiment, the concentration is 55% of saturation. In anotherembodiment, the concentration is 60% of saturation. In anotherembodiment, the concentration is 65% of saturation. In anotherembodiment, the concentration is 70% of saturation. In anotherembodiment, the concentration is 75% of saturation. In anotherembodiment, the concentration is 80% of saturation. In anotherembodiment, the concentration is 85% of saturation. In anotherembodiment, the concentration is 90% of saturation. In anotherembodiment, the concentration is 95% of saturation. In anotherembodiment, the concentration is 100% of saturation. In anotherembodiment, the concentration is near 100% of saturation. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment of methods and compositions of the presentinvention, the Listeria culture is flash-frozen in liquid nitrogen,followed by storage at the final freezing temperature. In anotherembodiment, the culture is frozen in a more gradual manner; e.g. byplacing in a vial of the culture in the final storage temperature. Inanother embodiment, the culture is frozen by any other method known inthe art for freezing a bacterial culture. Each possibility represents aseparate embodiment of the present invention.

In another embodiment of methods and compositions of the presentinvention, the storage temperature of the culture is between ⁻20 and ⁻80degrees Celsius (° C.). In another embodiment, the temperature issignificantly below ⁻20° C. In another embodiment, the temperature isnot warmer than ⁻70° C. In another embodiment, the temperature is ⁻70°C. In another embodiment, the temperature is about ⁻70° C. In anotherembodiment, the temperature is ⁻20° C. In another embodiment, thetemperature is about ⁻20° C. In another embodiment, the temperature is⁻30° C. In another embodiment, the temperature is ⁻40° C. In anotherembodiment, the temperature is ⁻50° C. In another embodiment, thetemperature is ⁻60° C. In another embodiment, the temperature is ⁻80° C.In another embodiment, the temperature is ⁻30-⁻70° C. In anotherembodiment, the temperature is ⁻40-⁻70° C. In another embodiment, thetemperature is ⁻50-⁻70° C. In another embodiment, the temperature is⁻60-⁻70° C. In another embodiment, the temperature is ⁻30-⁻80° C. Inanother embodiment, the temperature is ⁻40-⁻80° C. In anotherembodiment, the temperature is ⁻50-⁻80° C. In another embodiment, thetemperature is ⁻60-⁻80° C. In another embodiment, the temperature is⁻70-⁻80° C. In another embodiment, the temperature is colder than ⁻70°C. In another embodiment, the temperature is colder than ⁻80° C. Eachpossibility represents a separate embodiment of the present invention.

Methods for lyophilization and cryopreservation of recombinant Listeriastrains are well known to those skilled in the art. Each possibilityrepresents a separate embodiment of the present invention.

In one embodiment, “genetically fused” as provided herein is meant toresult in a chimeric DNA containing, each in its own discreteembodiment, a promoter and a coding sequence that are not associated innature.

In one embodiment, the “B-cell Receptors” or “BCR” is the cell-surfacereceptor of B cells for a specific antigen. In another embodiment, theBCR is composed of a transmembrane immunoglobulin molecule associatedwith the invariant Igα and Igβ chains in a noncovalent complex. Inanother embodiment, B-cell receptor (BCR) signaling regulates severalB-cell fate decisions throughout development. In another embodiment,continued expression of the signaling subunits of the BCR is requiredfor survival of mature B cells. In another embodiment, alterations inBCR signaling may support lymphomagenesis. In one embodiment, cells havethe CD20 protein on the outside of the cell. In another embodiment,cancerous B cells also carry the CD20 protein. In another embodiment,CD20 is highly expressed in at least 95% of B-cell lymphomas. In oneembodiment, the BCR is expressed in B-cell lymphomas. In anotherembodiment, the BCR is expressed in Follicular Lymphoma, SmallNon-Cleaved Cell Lymphoma, Marginal Zone Lymphoma, Splenic Lymphoma withvillous lymphocytes, Mantle Cell Lymphoma, Large Cell Lymphoma Diffuselarge Cell Lymphoma, Small Lymphocytic Lymphoma, Endemic Burkitt'slymphoma, Sporadic Burkitt's lymphoma, Non-Burkitt's lymphoma,Mucosa-Associated Lymphoid Tissue MALT/MALToma (extranodal), MonocytoidB-cell, lymphoma (nodal), Diffuse Mixed Cell, Immunoblastic Lymphoma,Primary Mediastinal B-Cell Lymphoma, Angiocentric Lymphoma—PulmonaryB-Cell. In another embodiment, CD20 is expressed in the BCR is expressedin Follicular Lymphoma, Small Non-Cleaved Cell Lymphoma, Marginal ZoneLymphoma, Splenic Lymphoma with villous lymphocytes, Mantle CellLymphoma, Large Cell Lymphoma Diffuse large Cell Lymphoma, SmallLymphocytic Lymphoma, Endemic Burkitt's lymphoma, Sporadic Burkitt'slymphoma, Non-Burkitt's lymphoma, Mucosa-Associated Lymphoid TissueMALT/MALToma (extranodal), Monocytoid B-cell, lymphoma (nodal), DiffuseMixed Cell, Primary Mediastinal B-Cell Lymphoma, AngiocentricLymphoma—Pulmonary B-Cell. Therefore, in one embodiment, thecompositions and methods of the present invention comprising BCR areparticularly useful in the prevention or treatment of theabove-mentioned cancers.

In one embodiment, a major etiological factor in the genesis of cervicalcarcinoma is the infection by human papillomaviruses (HPVs), which, inone embodiment, are small DNA viruses that infect epithelial cells ofeither the skin or mucosa. In one embodiment, HPV related malignanciesinclude oral, cervical, anogenital, and cervical cancers as well asrespiratory papillomatsis. In one embodiment, HPV expresses six or sevennon-structural proteins and two structural proteins, each of which mayserve as a target in the immunoprophylactic or immunotherapeuticapproaches described herein. In one embodiment, the viral capsidproteins L1 and L2 are late structural proteins. In one embodiment, L1is the major capsid protein, the amino acid sequence of which is highlyconserved among different HPV types.

In one embodiment, proteins E6 and E7 are two of seven earlynon-structural proteins, some of which play a role in virus replication(E1, E2, E4) and/or in virus maturation (E4). In another embodiment,proteins E6 and E7 are oncoproteins that are critical for viralreplication, as well as for host cell immortalization andtransformation. In one embodiment, E6 and E7 viral proteins are notexpressed in normal cervical squamous epithelia. In another embodiment,the expression of the E6 and E7 genes in epithelial stem cells of themucosa is required to initiate and maintain cervical carcinogenesis.Further and in some embodiments, the progression of pre-neoplasticlesions to invasive cervical cancers is associated with a continuousenhanced expression of the E6 and E7 oncoprotein. Thus, in anotherembodiment, E6 and E7 are expressed in cervical cancers. In anotherembodiment, the oncogenic potential of E6 and E7 may arise from theirbinding properties to host cell proteins. For example and in oneembodiment, E6 binds to the tumor-suppressor protein p53 leading toubiquitin-dependent degradation of the protein, and, in anotherembodiment, E7 binds and promotes degradation of the tumor-suppressorretinoblastoma protein (pRb). Therefore, in one embodiment, thecompositions and methods of the present invention comprising HPV-E7 areparticularly useful in the prevention or treatment of theabove-mentioned cancers.

NY-ESO-1 is, in one embodiment, a “cancer-testis” antigen expressed inepithelial ovarian cancer (EOC). In another embodiment, NY-ESO-1 isexpressed in metastatic melanoma, breast cancer, lung cancer, esophagealcancer, which in one embodiment, is esophageal squamous cell carcinoma,or a combination thereof. In one embodiment, NY-ESO-1 is one of the mostimmunogenic cancer testis antigens. In another embodiment NY-ESO-1 isable to induce strong humoral (antibody) and cellular (T cell) immuneresponses in patients with NY-ESO-1 expressing cancers either throughnatural or spontaneous induction by the patients tumor or followingspecific vaccination using defined peptide epitopes. In anotherembodiment, NY-ESO-1 peptide epitopes are presented by MHC class IImolecules Therefore, in one embodiment, the compositions and methods ofthe present invention comprising NY-ESO-1 are particularly useful in theprevention or treatment of the above-mentioned cancers.

EXPERIMENTAL DETAILS SECTION Example 1 LLO-Antigen Fusions InduceAnti-Tumor Immunity Materials and Experimental Methods(Examples 1-2)

Cell Lines

The C57BL/6 syngeneic TC-1 tumor was immortalized with HPV-16 E6 and E7and transformed with the c-Ha-ras oncogene. TC-1 expresses low levels ofE6 and E7 and is highly tumorigenic. TC-1 was grown in RPMI 1640, 10%FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 100μM nonessential amino acids, 1 mM sodium pyruvate, 50 micromolar (mcM)2-ME, 400 microgram (mcg)/ml G418, and 10% National Collection TypeCulture-109 medium at 37° with 10% CO₂. C3 is a mouse embryo cell fromC57BL/6 mice immortalized with the complete genome of HPV 16 andtransformed with pEJ-ras. EL-4/E7 is the thymoma EL-4 retrovirallytransduced with E7.

L. monocytogenes Strains and Propagation

Listeria strains used were Lm-LLO-E7 (hly-E7 fusion gene in an episomalexpression system; FIG. 1A), Lm-E7 (single-copy E7 gene cassetteintegrated into Listeria genome), Lm-LLO-NP (“DP-L2028”; hly-NP fusiongene in an episomal expression system), and Lm-Gag (“ZY-18”; single-copyHIV-1 Gag gene cassette integrated into the chromosome). E7 wasamplified by PCR using the primers 5′-GGCTCGAGCATGGAGATACACC-3′ (SEQ IDNO: 38; XhoI site is underlined) and 5′-GGGGACTAGTTTATGGTTTCTGAGAACA-3′(SEQ ID NO: 39; SpeI site is underlined) and ligated into pCR2.1(Invitrogen, San Diego, Calif.). E7 was excised from pCR2.1 by XhoI/SpeIdigestion and ligated into pGG-55. The hly-E7 fusion gene and thepluripotential transcription factor prfA were cloned into pAM401, amulticopy shuttle plasmid (Wirth R et al, J Bacteriol, 165: 831, 1986),generating pGG-55. The hly promoter drives the expression of the first441 AA of the hly gene product, (lacking the hemolytic C-terminus,referred to below as “ΔLLO,” and having the sequence set forth in SEQ IDNO: 17), which is joined by the XhoI site to the E7 gene, yielding ahly-E7 fusion gene that is transcribed and secreted as LLO-E7.Transformation of a prfA negative strain of Listeria, XFL-7 (provided byDr. Hao Shen, University of Pennsylvania), with pGG-55 selected for theretention of the plasmid in vivo (FIGS. 1A-B). The hly promoter and genefragment were generated using primers5′-GGGGGCTAGCCCTCCTTTGATTAGTATATTC-3′ (SEQ ID NO: 40; NheI site isunderlined) and 5′-CTCCCTCGAGATCATAATTTACTTCATC-3′ (SEQ ID NO: 41; XhoIsite is underlined). The prfA gene was PCR amplified using primers5′-GACTACAAGGACGATGACCGACAAGTGATAACCCGGGATCTAAATAAATCCGTT T-3′ (SEQ IDNO: 42; XbaI site is underlined) and 5′-CCCGTCGACCAGCTCTTCTTGGTGAAG-3′(SEQ ID NO: 43; SalI site is underlined). Lm-E7 was generated byintroducing an expression cassette containing the hly promoter andsignal sequence driving the expression and secretion of E7 into the orfZdomain of the LM genome. E7 was amplified by PCR using the primers5′-GCGGATCCCATGGAGATACACCTAC-3′ (SEQ ID NO: 44; BamHI site isunderlined) and 5′-GCTCTAGATTATGGTTTCTGAG-3′ (SEQ ID NO: 45; XbaI siteis underlined). E7 was then ligated into the pZY-21 shuttle vector. LMstrain 104035 was transformed with the resulting plasmid, pZY-21-E7,which includes an expression cassette inserted in the middle of a 1.6-kbsequence that corresponds to the orfX, Y, Z domain of the LM genome. Thehomology domain allows for insertion of the E7 gene cassette into theorfZ domain by homologous recombination. Clones were screened forintegration of the E7 gene cassette into the orfZ domain. Bacteria weregrown in brain heart infusion medium with (Lm-LLO-E7 and Lm-LLO-NP) orwithout (Lm-E7 and ZY-18) chloramphenicol (20 μg/ml). Bacteria werefrozen in aliquots at −80° C. Expression was verified by Westernblotting (FIG. 2)

Western Blotting

Listeria strains were grown in Luria-Bertoni medium at 37° C. and wereharvested at the same optical density measured at 600 nm Thesupernatants were TCA precipitated and resuspended in 1× sample buffersupplemented with 0.1 N NaOH. Identical amounts of each cell pellet oreach TCA-precipitated supernatant were loaded on 4-20% Tris-glycineSDS-PAGE gels (NOVEX, San Diego, Calif.). The gels were transferred topolyvinylidene difluoride and probed with an anti-E7 monoclonal antibody(mAb) (Zymed Laboratories, South San Francisco, Calif.), then incubatedwith HRP-conjugated anti-mouse secondary Ab (Amersham Pharmacia Biotech,Little Chalfont, U.K.), developed with Amersham ECL detection reagents,and exposed to Hyperfilm (Amersham Pharmacia Biotech).

Measurement of Tumor Growth

Tumors were measured every other day with calipers spanning the shortestand longest surface diameters. The mean of these two measurements wasplotted as the mean tumor diameter in millimeters against various timepoints. Mice were sacrificed when the tumor diameter reached 20 mm Tumormeasurements for each time point are shown only for surviving mice.

Effects of Listeria Recombinants on Established Tumor Growth

Six- to 8-wk-old C57BL/6 mice (Charles River) received 2×10⁵ TC-1 cellss.c. on the left flank. One week following tumor inoculation, the tumorshad reached a palpable size of 4-5 mm in diameter. Groups of eight micewere then treated with 0.1 LD₅₀ i.p. Lm-LLO-E7 (10⁷ CFU), Lm-E7 (10⁶CFU), Lm-LLO-NP (10⁷ CFU), or Lm-Gag (5×10⁵ CFU) on days 7 and 14.

⁵¹Cr Release Assay

C57BL/6 mice, 6-8 wk old, were immunized i.p. with 0.1 LD₅₀ Lm-LLO-E7,Lm-E7, Lm-LLO-NP, or Lm-Gag. Ten days post-immunization, spleens wereharvested. Splenocytes were established in culture with irradiated TC-1cells (100:1, splenocytes:TC-1) as feeder cells; stimulated in vitro for5 days, then used in a standard ⁵¹Cr release assay, using the followingtargets: EL-4, EL-4/E7, or EL-4 pulsed with E7 H-2b peptide (RAHYNIVTF;SEQ ID NO: 19). E:T cell ratios, performed in triplicate, were 80:1,40:1, 20:1, 10:1, 5:1, and 2.5:1. Following a 4-h incubation at 37° C.,cells were pelleted, and 50 μl supernatant was removed from each well.Samples were assayed with a Wallac 1450 scintillation counter(Gaithersburg, Md.). The percent specific lysis was determined as[(experimental counts per minute—spontaneous counts per minute)/(totalcounts per minute—spontaneous counts per minute)]×100.

TC-1-Specific Proliferation

C57BL/6 mice were immunized with 0.1 LD₅₀ and boosted by i.p. injection20 days later with 1 LD₅₀ Lm-LLO-E7, Lm-E7, Lm-LLO-NP, or Lm-Gag. Sixdays after boosting, spleens were harvested from immunized and naivemice. Splenocytes were established in culture at 5×10⁵/well inflat-bottom 96-well plates with 2.5×10⁴, 1.25×10⁴, 6×10³, or 3×10³irradiated TC-1 cells/well as a source of E7 Ag, or without TC-1 cellsor with 10 μg/ml Con A. Cells were pulsed 45 h later with 0.5 μCi[³H]thymidine/well. Plates were harvested 18 h later using a Tomtecharvester 96 (Orange, Conn.), and proliferation was assessed with aWallac 1450 scintillation counter. The change in counts per minute wascalculated as experimental counts per minute—no Ag counts per minute.

Flow Cytometric Analysis

C57BL/6 mice were immunized intravenously (i.v.) with 0.1 LD₅₀ Lm-LLO-E7or Lm-E7 and boosted 30 days later. Three-color flow cytometry for CD8(53-6.7, PE conjugated), CD62 ligand (CD62L; MEL-14, APC conjugated),and E7 H-2Db tetramer was performed using a FACSCalibur® flow cytometerwith CellQuest® software (Becton Dickinson, Mountain View, Calif.).Splenocytes harvested 5 days after the boost were stained at roomtemperature (rt) with H-2Db tetramers loaded with the E7 peptide(RAHYNIVTF; SEQ ID NO: 19) or a control (HIV-Gag) peptide. Tetramerswere used at a 1/200 dilution and were provided by Dr. Larry R. Pease(Mayo Clinic, Rochester, Minn.) and by the National Institute of Allergyand Infectious Diseases Tetramer Core Facility and the NationalInstitutes of Health AIDS Research and Reference Reagent Program.Tetramer⁺, CD8⁺, CD62L^(low) cells were analyzed.

Depletion of Specific Immune Components

CD8⁺ cells, CD4⁺ cells and IFN were depleted in TC-1-bearing mice byinjecting the mice with 0.5 mg per mouse of mAb: 2.43, GK1.5, or ×mg1.2,respectively, on days 6, 7, 8, 10, 12, and 14 post-tumor challenge. CD4⁺and CD8⁺ cell populations were reduced by 99% (flow cytometricanalysis). CD25⁺ cells were depleted by i.p. injection of 0.5 mg/mouseanti-CD25 mAb (PC61, provided by Andrew J. Caton) on days 4 and 6. TGFwas depleted by i.p. injection of the anti-TGF-mAb (2G7, provided by H.I. Levitsky), into TC-1-bearing mice on days 6, 7, 8, 10, 12, 14, 16,18, and 20. Mice were treated with 10⁷ Lm-LLO-E7 or Lm-E7 on day 7following tumor challenge.

Adoptive Transfer

Donor C57BL/6 mice were immunized and boosted 7 days later with 0.1 LD₅₀Lm-E7 or Lm-Gag. The donor splenocytes were harvested and passed overnylon wool columns to enrich for T cells. CD8⁺ T cells were depleted invitro by incubating with 0.1 μg 2.43 anti-CD8 mAb for 30 min at rt. Thelabeled cells were then treated with rabbit complement. The donorsplenocytes were >60% CD4⁺ T cells (flow cytometric analysis). TC-1tumor-bearing recipient mice were immunized with 0.1 LD₅₀ 7 dayspost-tumor challenge. CD4⁺-enriched donor splenocytes (10⁷) weretransferred 9 days after tumor challenge to recipient mice by i.v.injection.

B16F0-Ova Experiment

24 C57BL/6 mice were inoculated with 5×10⁵ B16F0-Ova cells. On days 3,10 and 17, groups of 8 mice were immunized with 0.1 LD₅₀ Lm-OVA (10⁶cfu), Lm-LLO-OVA (10⁸ cfu) and eight animals were left untreated.

Statistics

For comparisons of tumor diameters, mean and SD of tumor size for eachgroup were determined, and statistical significance was determined byStudent's t test. p<0.05 was considered significant.

Results

Lm-E7 and Lm-LLO-E7 were compared for their abilities to impact on TC-1growth. Subcutaneous tumors were established on the left flank ofC57BL/6 mice. Seven days later tumors had reached a palpable size (4-5mm) Mice were vaccinated on days 7 and 14 with 0.1 LD₅₀ Lm-E7,Lm-LLO-E7, or, as controls, Lm-Gag and Lm-LLO-NP. Lm-LLO-E7 inducedcomplete regression of 75% of established TC-1 tumors, while the other 2mice in the group controlled their tumor growth (FIG. 3A). By contrast,immunization Lm-E7 and Lm-Gag did not induce tumor regression. Thisexperiment was repeated multiple times, always with very similarresults. In addition, similar results were achieved for Lm-LLO-E7 underdifferent immunization protocols. In another experiment, a singleimmunization was able to cure mice of established 5 mm TC-1 tumors.

In other experiments, similar results were obtained with two otherE7-expressing tumor cell lines: C3 and EL-4/E7. To confirm the efficacyof vaccination with Lm-LLO-E7, animals that had eliminated their tumorswere re-challenged with TC-1 or EL-4/E7 tumor cells on day 60 or day 40,respectively. Animals immunized with Lm-LLO-E7 remained tumor free untiltermination of the experiment (day 124 in the case of TC-1 and day 54for EL-4/E7).

A similar experiment was performed with the chicken ovalbumin antigen(OVA). Mice were immunized with either Lm-OVA or Lm-LLO-OVA, thenchallenged with either an EL-4 thymoma engineered to express OVA or thevery aggressive murine melanoma cell line B16F0-Ova, which has very lowMHC class I expression. In both cases, Lm-LLO-OVA, but not Lm-OVA,induced the regression of established tumors. For example, at the end ofthe B16F0 experiment (day 25), all the mice in the naive group and theLm-OVA group had died. All the Lm-LLO-OVA mice were alive, and 50% ofthem were tumor free. (FIG. 3B).

Thus, expression of an antigen gene as a fusion protein with ΔLLOenhances the immunogenicity of the antigen.

Example 2 LM-LLO-E7 Treatment Elicits TC-1 Specific SplenocyteProliferation

To measure induction of T cells by Lm-E7 with Lm-LLO-E7, TC-1-specificproliferative responses of splenocytes from rLm-immunized mice, ameasure of antigen-specific immunocompetence, were assessed. Splenocytesfrom Lm-LLO-E7-immunized mice proliferated when exposed to irradiatedTC-1 cells as a source of E7, at splenocyte: TC-1 ratios of 20:1, 40:1,80:1, and 160:1 (FIG. 4). Conversely, splenocytes from Lm-E7 and rLmcontrol immunized mice exhibited only background levels ofproliferation.

Example 3 Fusion of NP to LLO Enhances its Immunogenicity Materials andExperimental Methods

Lm-LLO-NP was prepared as depicted in FIG. 1, except that influenzanucleoprotein (NP) replaced E7 as the antigen. 32 BALB/c mice wereinoculated with 5×10⁵ RENCA-NP tumor cells. RENCA-NP is a renal cellcarcinoma retrovirally transduced with influenza nucleoprotein NP(described in U.S. Pat. No. 5,830,702, which is incorporated herein byreference). After palpable macroscopic tumors had grown on day 10, eightanimals in each group were immunized i.p. with 0.1 LD₅₀ of therespective Listeria vector. The animals received a second immunizationone week later.

Results

In order to confirm the generality of the finding that fusing LLO to anantigen confers enhanced immunity, Lm-LLO-NP and Lm-NP (similar to theLm-E7 vectors) were constructed, and the vectors were compared forability to induce tumor regression, with Lm-Gag (isogenic with Lm-NPexcept for the antigen expressed) as a negative control. As depicted inFIG. 5, 6/8 of the mice that received Lm-LLO-NP were tumor free. Bycontrast, only 1/8 and 2/8 mice in the Lm-Gag and Lm-NP groups,respectively, were tumor free. All the mice in the naive group had largetumors or had died by day 40. Thus, enhancement of immunogenicity of anantigen by fusion to LLO is not restricted to E7, but rather is ageneral phenomenon.

Example 4 Enhancement of Immunogenicity by Fusion of an Antigen to LLOdoes not Require a Listeria Vector Materials and Experimental Methods

Construction of Vac-SigE7Lamp

The WR strain of vaccinia was used as the recipient and the fusion genewas excised from the Listeria plasmid and inserted into pSC11 under thecontrol of the p75 promoter. This vector was chosen because it is thetransfer vector used for the vaccinia constructs Vac-SigE7Lamp andVac-E7 and would therefore allow direct comparison with Vac-LLO-E7. Inthis way all three vaccinia recombinants would be expressed undercontrol of the same early/late compound promoter p7.5. In addition, SC11allows the selection of recombinant viral plaques to TK selection andbeta-galactosidase screening. FIG. 6 depicts the various vacciniaconstructs used in these experiments. Vac-SigE7Lamp is a recombinantvaccinia virus that expressed the E7 protein fused between lysosomalassociated membrane protein (LAMP-1) signal sequence and sequence fromthe cytoplasmic tail of LAMP-1. It was designed to facilitate thetargeting of the antigen to the MHC class II pathway.

The following modifications were made to allow expression of the geneproduct by vaccinia: (a) the T5XT sequence that prevents earlytranscription by vaccinia was removed from the 5′ portion of the LLO-E7sequence by PCR; and (b) an additional XmaI restriction site wasintroduced by PCR to allow the final insertion of LLO-E7 into SC11.Successful introduction of these changes (without loss of the originalsequence that encodes for LLO-E7) was verified by sequencing. Theresultant pSCl 1-E7 construct was used to transfect the TK-ve cell lineCV1 that had been infected with the wild-type vaccinia strain, WR. Celllysates obtained from this co-infection/transfection step containvaccinia recombinants that were plaque-purified 3 times. Expression ofthe LLO-E7 fusion product by plaque purified vaccinia was verified byWestern blot using an antibody directed against the LLO proteinsequence. In addition, the ability of Vac-LLO-E7 to produce CD8⁺ T cellsspecific to LLO and E7 was determined using the LLO (91-99) and E7(49-57) epitopes of Balb/c and C57/BL6 mice, respectively. Results wereconfirmed in a chromium release assay.

Results

To determine whether enhancement of immunogenicity by fusion of anantigen to LLO requires a Listeria vector, a vaccinia vector expressingE7 as a fusion protein with a non-hemolytic truncated form of LLO (ΔLLO)was constructed. Tumor rejection studies were performed with TC-1following the protocol described for Example 1. Two experiments wereperformed with differing delays before treatment was started. In oneexperiment, treatments were initiated when the tumors were about 3 mm indiameter (FIG. 7). As of day 76, 50% of the Vac-LLO-E7 treated mice weretumor free, while only 25% of the Vac-SigE7Lamp mice were tumor free. Inother experiments, ΔLLO-antigen fusions were more immunogenic than E7peptide mixed with SBAS2 or unmethylated CpG oligonucleotides in aside-by-side comparison.

These results show that (a) fusion of ΔLLO-antigen fusions areimmunogenic not only in the context of Listeria, but also in othercontexts; and (b) the immunogenicity of ΔLLO-antigen fusions comparesfavorably with other accepted vaccine approaches.

Example 5 Site-Directed Mutagenesis of the LLO Cholesterol-BindingDomain

Site-directed mutagenesis was performed on LLO to introduce inactivatingpoint mutations in the CBD, using the following strategy. The resultingprotein is termed “mutLLO”:

Subcloning of LLO into pET29b

The amino acid sequence of wild-type LLO is:

MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWE

RTVIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIE (SEQ ID NO: 46). The signalpeptide and the cholesterol-binding domain (CBD) are underlined, with 3critical residues in the CBD (C484, W491, and W492) in bold-italics.

A 6×His tag (HHHHHH) was added to the C-terminal region of LLO. Theamino acid sequence of His-tagged LLO is:

(SEQ ID NO: 47) MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIEHHHHHH.

A gene encoding a His-tagged LLO protein was digested with NdeI/BamHI,and the NdeI/BamHI was subcloned into the expression vector pET29b,between the NdeI and BamHI sites. The sequence of the gene encoding theLLO protein is:

catatgaaggatgcatctgcattcaataaagaaaattcaatttcatccgtggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaatcgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgccgccaagaaaaggttacaaagatggaaatgaatatattgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacattcaagttgtgaatgcaatttcgagcctaacctatccaggtgctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgtaaaacgtgattcattaacactcagcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacgttaacaacgcagtaaatacattagtggaaagatggaatgaaaaatatgctcaagcttattcaaatgtaagtgcaaaaattgattatgatgacgaaatggcttacagtgaatcacaattaattgcgaaatttggtacagcatttaaagctgtaaataatagcttgaatgtaaacttcggcgcaatcagtgaagggaaaatgcaagaagaagtcattagttttaaacaaatttactataacgtgaatgttaatgaacctacaagaccttccagatttttcggcaaagctgttactaaagagcagttgcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatggccgtcaagtttatttgaaattatcaactaattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcaggtgatgtagaactaacaaatatcatcaaaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcgacggcaacctcggagacttacgcgatattttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgcttatacaacaaacttcctaaaagacaatgaattagctgttattaaaaacaactcagaatatattgaaacaacttcaaaagcttatacagatggaaaaattaacatcgatcactctggaggatacgttgctcaattcaacatttcttgggatgaagtaaattatgatcctgaaggtaacgaaattgttcaacataaaaactggagcgaaaacaataaaagcaagctagctcatttcacatcgtccatctatttgcctggtaacgcgagaaatattaatgtttacgctaaagaatgcactggtttagcttgggaatggtggagaacggtaattgatgaccggaacttaccacttgtgaaaaatagaaatatctccatctggggcaccacgctttatccgaaatatagtaataaagtagataatccaatcgaacaccaccaccaccaccactaataaggatcc(SEQ ID NO: 48). The underlined sequences are, starting from thebeginning of the sequence, the NdeI site, the NheI site, theCBG-encoding region, the 6×His tag, and the BamHI site. The CBD residesto be mutated in the next step are in bold-italics.

Splicing by Overlap Extension (SOE) PCR

Step 1: PCR reactions #1 and #2 were performed on the pET29b-LLOtemplate. PCR reaction #1, utilizing primers #1 and #2, amplified thefragment between the NheI site and the CBD, inclusive, introducing amutation into the CBD. PCR reaction #2, utilizing primers #3 and #4,amplified the fragment between the CBD and the BamHI site, inclusive,introducing the same mutation into the CBD (FIG. 8A).

PCR reaction #1 cycle: A) 94° C. 2 min 30 sec, B) 94° C. 30 sec, C) 55°C. 30 sec, D) 72° C. 1 min, Repeat steps B to D 29 times (30 cyclestotal), E) 72° C. 10 min

PCR reaction #2 cycle: A) 94° C. 2 min 30 sec, B) 94° C. 30 sec, C) 60°C. 30 sec, D) 72° C. 1 min, Repeat steps B to D 29 times (30 cyclestotal), E) 72° C. 10 min

Step 2: The products of PCR reactions #1 and #2 were mixed, allowed toanneal (at the mutated CBD-encoding region), and PCR was performed withprimers #1 and #4 for 25 more cycles (FIG. 8B). PCR reaction cycle: A)94° C. 2 min 30 sec, B) 94° C. 30 sec, C) 72° C. 1 min, Repeat steps Bto C 9 times (10 cycles total), Add primers #1 and #4, D) 94° C. 30 sec,E) 55° C. 30 sec, F) 72° C. 1 min, Repeat steps D to F 24 times (25cycles total), G) 72° C. 10 min

Primer sequences:

Primer 1 (SEQ ID NO: 49; NheI sequence is underlined)GCTAGCTCATTTCACATCGT Primer 2:(SEQ ID NO: 50; CBD-encoding sequence is under-lined; mutated codons are in bold-italics) TCT

TTCCCAAGCTAAACCAGT

TTCTTTAGCGTAAACAT TAATATT. Primer 3:(SEQ ID NO: 51; CBD-encoding sequence is under-lined; mutated codons are in bold-italics) GAA

ACTGGTTTAGCTTGGGAA

AGAACGGTAATTGATGA CCGGAAC. Primer 4:(SEQ ID NO: 52; BamHI sequence is underlined)GGATCCTTATTAGTGGTGGTGGTGGTGGTGTTCGATTGG.

The wild-type CBD sequence is ECTGLAWEWWR (SEQ ID NO: 18).

The mutated CBD sequence is EATGLAWEAAR (SEQ ID NO: 53).

The sequence of the mutated NheI-BamHI fragment is

(SEQ ID NO: 54) GCTAGCTCATTTCACATCGTCCATCTATTTGCCTGGTAACGCGAGAAATATTAATGTTTACGCTAAAGAA

ACTGGTTTAGCTTGGGAA

AGA ACGGTAATTGATGACCGGAACTTACCACTTGTGAAAAATAGAAATATCTCCATCTGGGGCACCACGCTTTATCCGAAATATAGTAATAAAGTAGATAATCCAATCGAACACCACCACCACCACCACTAATAAGGATCC.

Example 6 Replacement of Part of the LLO CBD with a CTL Epitope

Site-directed mutagenesis was performed on LLO to replace 9 amino acids(AA) of the CBD with a CTL epitope from the antigen NY-ESO-1. Thesequence of the CBD (SEQ ID NO: 18) was replaced with the sequenceESLLMWITQCR (SEQ ID NO: 55; mutated residues underlined), which containsthe HLA-A2 restricted epitope 157-165 from NY-ESO-1, termed “ctLLO.”

The subcloning strategy used was similar to the previous Example.

The primers used were as follows:

Primer 1: (SEQ ID NO: 56; NheI sequence is underlined)GCTAGCTCATTTCACATCGT. Primer 2:(SEQ ID NO: 57; CBD-encoding sequence is under-lined; mutated (NY-ESO-1) codons are in bold- italics) TCT

TTCTTTAGCGTAAACATTAA TATT. Primer 3:(SEQ ID NO: 58; CBD-encoding sequence is underlined;mutated (NY-ESO-1) codons are in bold-italics) GAA

AGAACGGTAATTGATGACCG GAAC. Primer 4:(SEQ ID NO: 59; BamHI sequence is underlined)GGATCCTTATTAGTGGTGGTGGTGGTGGTGTTCGATTGG.

The sequence of the resulting NheI/BamHI fragment is as follows:

(SEQ ID NO: 60) GCTAGCTCATTTCACATCGTCCATCTATTTGCCTGGTAACGCGAGAAATATTAATGTTTACGCTAAAGAA

AGA ACGGTAATTGATGACCGGAACTTACCACTTGTGAAAAATAGAAATATCTCCATCTGGGGCACCACGCTTTATCCGAAATATAGTAATAAAGTAGATAATCCAATCGAACACCACCACCACCACCACTAATAAGGATCC.

Example 7 mutLLO and ctLLO are Able to be Expressed and Purified in E.coli Expression Systems

To show that mutLLO and ctLLO could be expressed in E. coli, E. coliwere transformed with pET29b and induced with 0.5 mM IPTG, then celllysates were harvested 4 hours later and the total proteins wereseparated in a SDS-PAGE gel and subject to Coomassie staining (FIG. 9A)and anti-LLO Western blot, using monoclonal antibody B3-19 (FIG. 9B).Thus, LLO proteins containing point mutations or substitutions in theCBD can be expressed and purified in E. coli expression systems.

Example 8 mutLLO AND ctLLO Exhibit Significant Reduction in HemolyticActivity Materials and Experimental Methods

Hemolysis Assay

1. Wild-type and mutated LLO were diluted to the dilutions indicated inFIGS. 10A-B in 900 μl of 1×PBS-cysteine (PBS adjusted to pH 5.5 with 0.5M Cysteine hydrochloride or was adjusted to 7.4). 2. LLO was activatedby incubating at 37° C. for 30 minutes. 3. Sheep red blood cells (200μl/sample) were washed twice in PBS-cysteine and 3 to 5 times in 1×PBSuntil the supernatant was relatively clear. 4. The final pellet of sheepred blood cells was resuspended in PBS-cysteine and 100 μl of the cellsuspension was added to the 900 μl of the LLO solution (10% finalsolution). 5. 50 μl of sheep red blood cells was added to 950 μl ofwater+10% Tween 20 (Positive control for lysis, will contain 50% theamount of lysed cells as the total amount of cells add to the othertubes; “50% control.”) 6. All tubes were mixed gently and incubated at37° C. for 45 minutes. 7. Red blood cells were centrifuged in amicrocentrifuge for 10 minutes at 1500 rpm. 8. A 200 μl aliquot of thesupernatant was transferred to 96-well ELISA plate and read at 570 nm tomeasure the concentration of released hemoglobin after hemolysis, andsamples were titered according to the 50% control.

Results

The hemolytic activity of mutLLO and ctLLO was determined using a sheepred blood cell assay. mutLLO exhibited significantly reduced (between100-fold and 1000-fold) hemolytic titer at pH 5.5, and undetectablehemolytic activity at pH 7.4. ctLLO exhibited undetectable hemolyticactivity at either pH (FIGS. 10A-B).

Thus, point (mutLLO) or substitution (ctLLO) mutation of LLO CBDresidues, including C484, W491, and W492, abolishes or severely reduceshemolytic activity. Further, replacement of the CBD with a heterologousantigenic peptide is an effective means of creating an immunogeniccarrier of a heterologous epitope, with significantly reduced hemolyticactivity relative to wild-type LLO.

Example 9 Expression of the 38C13 BCR as an scFv Protein

A modified pUC119 plasmid was utilized to express the scFv protein in E.coli (Sure® strain, Statagene, La Jolla, Calif.). The plasmid containedthe 38C13 scFv DNA (provided by Dr. Ronald Levy, Stanford University),sequences coding for the bacterial leader pelB (facilitates secretion ofthe protein into the periplasmic space) and the human c-myc peptide tag,which aids detection of protein expression in E. coli and purificationof the tumor antigen. The 38C13 VH sequence starts with the Gly residueencoded by residues 133-135 and ends with the Val residue encoded byresidues 478-480. The 38C13 VK sequence starts with the Glu residueencoded by residues 538-540). The 38C13 VK has a myc tag on the end; theVK ends with a Lys (encoded by residues 848-850).

The relevant fragment of the plasmid had the following sequence:

(SEQ ID NO: 61) gcccagccgccatgccaggtgaagctgcaggagtcaggaggaggcttggtccagcctgggggttctctgagtctctcctgtgcagcttctggattcaccttcactgattactacatgagctgggtccgccagcctccagggaaggcacttgagtggttggctttgattagaaacaaagctaatggttacacagagtacagtgcatctgtgaagggtcggttcaccatctccagagataattcccaaagcatcctctatcttcaaatgaatgccctgagagctgaggacagtgccacttattactgtgcaagagatcccaattactacgatggtagctacgaagggtactttgactactggggccaagggaccacggtcaccgtctcctcaggcggaggcggttcaggcggaggtggctctggcggtggcggatcggacattgagctcacccagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggcaagccaagacattaacaagtatatagcttggtaccaacacaagcctggaaaaggtcctaggctgctcatacattacacatctacattacagccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccttcagcatcagcaacctggagcctgaagatattgcaacttattattgtctacagtatgataatctgtacacgttcggctcggggaccaagctggaaataaaacgggcggccgcagaacaaaaactcatctcagaagaggatctgaatta ataagaattc.

The encoded protein had the sequence:

(SEQ ID NO: 62) MKYLLPTAAAGLLLLAAQPAQPPCQVKLQESGGGLVQPGGSLSLSCAASGFTFTDYYMSWVRQPPGKALEWLALIRNKANGYTEYSASVKGRFTISRDNSQSILYLQMNALRAEDSATYYCARDPNYYDGSYEGYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLYTFGSGTKLEIKRAAAEQKLISEEDLN.

Initially, the 38C13 plasmid was transformed into the E. coli strainBL21*. Following IPTG-induction, the BL21* cells expressed therecombinant protein, with a minor fraction present in the periplasmicspace, and the majority present in the E. coli inclusion bodies. Theinclusion bodies were solubilized (at <80 mcg/ml total protein) in 6Mguanidine; the solubilized proteins were refolded in the presence ofL-arginine, oxidized glutathione, and EDTA at 10° C. for 3-5 days. Therefolded 38scFv protein was then purified from other proteins on animmuno-affinity column containing the S1C5 antibody (anti-38C13 BCRclone) linked to CNBr sepharose using the Amino-link® kit (PierceEndogen).

To increase the yield, recombinant protein was recovered from solubleprotein extracts. Induction of 38C13scFv expression and recovery ofsoluble versus insoluble protein at 20° C. and 30° C. were compared.Greater yields of soluble 38C13scFv were recovered by induction at 20°C. Furthermore, maximal yield of soluble protein in the culturesupernatant (SN) or from cells was achieved when 0.5% glycine or 1%TX-100 was included in the induction medium. Finally, a 1-literinduction culture performed in medium containing 0.5% glycine and 1%TX-100 yielded 2.34 mg pure soluble 38C13scFv following affinitychromatography (FIG. 11). The overall strategy for production of scFVtumor antigen is summarized in FIG. 12.

Example 10 Verification of 38C13 SCFV Conformational Integrity by ELISA

To verify that correctly folded 38C13scFv protein was produced by theabove method, an ELISA assay was developed. Using serial dilutions ofpurified 38C13scFv protein, a standard curve was established. This assayshowed that correctly folded 38C13scFv protein was produced (FIG. 13).This ELISA assay can also be used to quantitate correctly folded solubleprotein in induction media as well as cell protein extracts, for furtheroptimization of conditions for producing 38C13scFv protein.

Production of Non-Hemolytic LLO: LLO was rendered non-hemolytic byconjugation to 38C13 BCR with aldehyde. The E. coli LLO/pET29 plasmidwas obtained from Dr. Margaret Gedde (Gedde M M, Higgins D E, Tilney LG, Portnoy D A. Role of listeriolysin O in cell-to-cell spread ofListeria monocytogenes. Infect Immun 2000 February; 68(2):999-1003)which has a (HIS)₆ tag at the 3′ end The E. coli strain BL21* wastransformed with LLO/pET29 plasmid by temperature shock and the bacteriaplated onto selection medium. Colonies were selected and theBL21*/LLO/pet29 were cultured in LB medium at 37° C. in an incubatorshaker until plateau phase. Cell aliquots were then frozen inLB/glycerol at −20° C. When induction expression was to be performed,the frozen BL21*/LLO/pet29 were streaked onto agar selection plates,plates were incubated overnight at 37° C., colonies selected and grownin LB broth with kanamycin and chloramphenicol. The culture was thenincubated at 30° C. in an incubator shaker until the OD₆₀₀=0.6-0.7, atthis point IPTG (1 mM final concentration) was added to the culture andincubated for a further 8 hours. The LB broth was then spun at 5,000rpm, the cells were collected and frozen at −20° C. until affinitypurification was to be performed. Recombinant LLO (AA sequence 20 to 442of LLO, excluding the signal sequence) was purified from the bacteriasoluble protein fraction according to the protocol provided by QIAGEN.Briefly, frozen cells were thawed on ice, incubated in lysis buffer (50mM phosphate, pH8, 500 mM NaCl, 20 mM imidazole, 10 mMβ-mercaptoethanol) supplemented with lysozyme (Calbiochem) and acocktail of protease inhibitors (Roche) for 30 minutes at 4° C. Thecells were then sonicated three times for 10 second bursts each untilall bacterial clumps were removed. The lysed bacteria were then spun at24,000 g and the supernatant removed (soluble protein fraction). Thesoluble proteins were then loaded onto a pre-equilibrated Ni⁺-NTAagarose (QIAGEN) column; weakly bound proteins were removed by washinguntil the A₂₈₀=<0.05. At this point, the recombinant LLO was removedfrom the column by addition of elution buffer containing 500 mMimidazole. Elution fractions were collected and pooled, dialyzed againstLLO storage buffer overnight (50 mM phosphate/acetate, pH6, 1M NaCl, 5mM DTT) at 4° C. and then stored in 1 mg/ml aliquots at −80° C. untilneeded.

Conjugation of 38C13 Lymphoma Idiotype Protein to Immunogenic Proteinsand Validation

38C13 Id protein was conjugated to either Keyhole Limpet Hemocyanin(KLH, Pierce Endogen) or purified recombinant LLO using glutareldehydeto cross-link primary amines on the proteins. Briefly, 1 mg/1 Id proteinand 1 mg/ml immunogen were combined in a sterile tube with fresh 0.1%glutaraldehyde (SIGMA), the proteins were then mixed on a rotator for10-15 minutes at room temperature. The conjugated proteins were thendialyzed against 0.1M PBS at 4° C. overnight; the conjugation of Idprotein to immunogens was confirmed complete by SDS-PAGE and Coumassiestain. The conjugate proteins were then depleted of endotoxin usingDetoxigel (Pierce Endogen), all vaccines had <1 EU/μg protein. Proteinconjugates containing recombinant LLO were checked for the absence ofresidual lytic function using sheep red cells as the target as describedfor detoxLLO.

Example 11 Construction of the 38C13 BCR-LLO Vaccine

Purification of idiotype proteins. B-cell lymphoma idiotype proteinswere purified from hybridoma supernatant via differential ammoniumsulfate precipitation. The process for production of the 38C13 lymphomaidiotype protein is outlined in FIG. 14. The 38C13A1.2 hybridomasecreted the IgM protein into the Bioreactor (BD Celline) supernatant.The IgM protein was recovered from the bioreactor supernatant followingdifferential ammonium sulfate precipitation. Samples from each fractionwere run by SDS-PAGE under reducing and non-reducing conditions andcharacterized by Coumassie stain (see FIG. 15). The 45% fraction fromthe bioreactor supernatant contained the 38C13 IgM protein; recovery was2 mg/ml supernatant.

Recombinant LLO was recovered from soluble proteins from BL21* followingIPTG-induced expression induction for 18 hours at 30° C. The solubleproteins were incubated in batch form with Ni⁺-NTA agarose for 30minutes at room temperature. Non-specifically bound proteins wereremoved following a washing step in phosphate buffer, pH 8 containing 20mM imidazole. The recombinant LLO-His was then eluted from the columnusing phosphate buffer pH 8 plus 500 mM imidazole. The purity of theelution fractions was confirmed by SDS PAGE followed by Coumassie stainor Western blot using the Mab B3-19. Results show (FIG. 16) that asingle band of molecular weight 58 kD was eluted from the Ni⁺-NTA columnand its identity as confirmed as LLO by the Mab B3-19.

Subsequently, the 38C13 idiotype protein was conjugated to recombinantLLO or KLH using 0.1% glutaraldehyde for 10 minutes at room temperature.The glutaraldehyde was removed following dialysis against 0.1M PBS at 4°C. overnight. The 38Id-LLO and 38Id-KLH conjugates were thencharacterized by SDS-PAGE under reducing and non-reducing conditionsfollowed by Coumassie stain (FIG. 17). Results showed the conjugationwas successful with no free 38Id protein nor immunogenic protein ineither conjugate. To ascertain that the 38C13 idiotype epitope was stillpresent following conjugation, a FACS-based competitive binding assaywas developed (FIG. 18). This assay detects the ability of the 38Idconjugate to block the specific binding of FITC conjugated S1C5 Mab tothe 38C13 lymphoma BCR. The presence of 100 ng 38Id protein wassufficient to block binding of the 0.1 ug S1C5 Mab to 38C13 lymphomacells (FIG. 19). In contrast, 1 mcg 38Id-LLO or 10 mcg 38Id-KLH wererequired to block binding of 0.1 mcg S1C5 Mab to 38C13 lymphoma cells.

Example 12 38C13 BCR-LLO Vaccines are Efficacious in a MouseNon-Hodgkin's Lymphoma Tumor Protection Model

C3H/HeN mice (n=8) were vaccinated with (a) 38C13 idiotype protein(38-Id), (b) 38Id coupled to Keyhole Limpet Hemocyanin (38Id-KLH); (c)38Id-LLO; or (d) PBS (negative control). Vaccines were administered astwo 50 mcg s.c. doses on days 0 and 14 days, with 10,000 U murine GMCSF(mGM-CSF). In addition, 10,000 U Mgm-CSF was administered on the sameflank for 3 consecutive days. On day 28, mice were challenged with 10³38 C13 lymphoma cells on the flank used for immunization, and tumorformation was monitored for 100 days. The 38Id-LLO vaccine induced tumorprotection in 7/8 mice up to 65 days (FIG. 20). Mice vaccinated with38Id-KLH were shown to have a lower level of resistance to the 38C13lymphoma (5/8 tumor free at day 65) compared to mice vaccinated with38Id-LLO (7/8 tumor free), but this did not reach statisticalsignificance (p=0.273). In contrast, mice immunized with 38Id or PBS hadpoor 38C13 lymphoma resistance, with all mice developing tumors by day22. When the incidence of tumor formation was examined statistically,the test vaccine group (Id-LLO) was shown to have a significantly lowerincidence of tumors versus the 38Id (p=0.0016) or the PBS group(p=0.0001).

Example 13 38Id-LLO Induces High Titer Anti-Idiotype Antibodies afterOne Immunization with a Strong Ig2A Subtype

Peripheral blood samples were collected from individual mice prior toand 12 days after each immunization. The serum samples were then testedby ELISA assay for the presence of anti-idiotype antibodies. Mice fromthe 38Id-LLO and 38Id-KLH vaccine groups were the only vaccine groupswith sera positive for anti-idiotype antibodies (FIG. 21A). Comparedwith control vaccine groups, mice immunized with 38Id-LLO had high titeranti-idiotype antibodies following one immunization; whereas miceimmunized with 38Id-KLH required two immunizations to achieve the sametiter. An isotyping assay was performed to characterize theanti-idiotype antibodies induced by 38Id-LLO versus 38Id-KLH. Followinga single immunization with 38Id-LLO, a high titer polyclonal responsewas induced with equivalent levels of IgG1 and IgG2a anti-idioypeantibodies (FIG. 21B). The level of the 38Id-LLO induced antibodiesincreased after the second immunization; however the ratio of IgG1:IgG2a(1.0) remained the same. In contrast, the 38Id-KLH vaccine induced ahigher level of IgG1 versus IgG2a anti-idiotype antibodies after bothimmunizations (IgG1:IgG2a ratio was 1.8 and 1.3 respectively; FIG. 21B).Levels of IgG2a anti-Id antibodies were statistically different betweenId-LLO and Id-KLH sera after the first (p=0.0001) and second (p=0.002)immunizations. The level of IgG1 anti-idiotype antibody was onlystatistically different between these two vaccine groups after the firstimmunization (p=0.03). The anti-Id antibody status correlated well withthe days to formation of a tumor for each vaccine group. While the naïveand Id alone vaccine groups had all formed tumors by day 18 and werenegative for anti-Id antibodies, all Id-LLO and Id-KLH immunized micedeveloped anti-Id antibodies, and this correlated with tumor resistance,with 7/8 Id-LLO mice and 5/8 Id-KLH mice tumor free 60 days after 38C13challenge.

To confirm the above results, the ability of immunized mouse serum toblock binding of S1C5-FITC to 38C13 cells was measured, as a decrease influorescence by FACS. In the first experiment (FIG. 22A), the bindingspecificity of S1C5 to the 38C13 lymphoma idiotype was verified.Subsequently, the inhibition of S1C5 binding to 38C13 cells by mouseserum (taken at various stages through Id-LLO immunization and aftertumor challenges) was investigated (FIG. 22B). In this study, mouseserum inhibited binding of S1C5 to the 38C13 cells after the 1^(st) and2^(nd) immunizations and after tumor challenges, but notpre-immunization

Example 14 38Id-LLO Immunization Induces a Th1 Response in the DLN

To investigate the CD4⁺ and CD8⁺ T cell responses to the proteinvaccines, DLN were harvested 14 days after the immunization protocol.Cytokine secretion was examined by FACS, CD4⁺ T cells from the Id-LLOvaccine group secreted IFN-γ in response to in vitro re-stimulation withLLO protein versus the PBS control (p=0.02, FIG. 23A). In no othervaccine groups did significant numbers of CD4⁺ T cells secrete IFN-γ inresponse to protein re-stimulation. However, CD4⁺ T cells from the samevaccine groups did not respond significantly to in vitro re-stimulationwith 38Id protein.

IL-4 secretion in response to in vitro protein re-stimulation was alsoexamined. In mice immunized with Id-KLH, Id-LLO or LLO alone, CD4⁺ DLNcells responded significantly to in vitro restimulation with theimmunogens KLH or LLO (FIG. 23B). The frequency of CD4 T cells (from theId-LLO vaccine groups) responding to LLO re-stimulation by secretingIL-4 (0.7)% was lower than that observed for CD4 T cells secreting IFN-γ(3.8)%. Simultaneously, DLN CD8 T cell response to proteinre-stimulation was examined in immunized mice (FIG. 23C). The level ofIFN-γ secretion in response to LLO re-stimulation was 18% in miceimmunized with Id-LLO (p=0.0005) significantly higher compared to KLHre-stimulated Id-KLH immunized mice (10%, p=0.009). A significantresponse to re-stimulation with the idiotype protein was also seen inthe DLN CD8 T cells from mice immunized with Id-LLO (p=0.04) and Id-KLH(p=0.02).

Proliferative responses of DLN CD4 T cells to immunization and in vitrore-stimulation were examined by CFSE fluorescence. In each vaccinegroup, the conA positive control demonstrated the proliferativepotential of the cells (FIG. 24). This proliferative response wascharacterized by 7 cell divisions by a subset of the initial CD4 T cellpopulation. The average number of cell divisions responding CD4 T cellsunderwent in response to in vitro re-stimulation (proliferative index)ranged from 1.7-3.0 in the presence of conA (FIGS. 23D and 24). Therewas no proliferation in the 2 control vaccine groups, PBS and 38Id, inthe presence or absence of protein re-stimulation (proliferative indexof 1.0), while cells from the 38Id-LLO and 38Id-KLH vaccine groupsresponded to 38Id protein restimulation with a proliferative index of1.2 in each vaccine group. Also, the Id-LLO vaccine group cellsexhibited a marked proliferative capacity in response to LLOre-stimulation (proliferative index of 3.2). The response of cells fromthe 38Id-KLH group to KLH re-stimulation had a proliferative index of1.3.

Thus, 38Id-LLO immunization induces a draining lymph node Th1 response.

Example 15 Construction and Testing of mutLLO-38C13 BCR and ctLLO-38C13BCR Vaccines

mutLLO-38C13 BCR and ctLLO-38C13 BCR vaccines are constructed frommutLLO-, ctLLO-, and 38C13-encoding DNA as described in Example 11. Thevaccines are tested as described in Example 12, and are found to exhibitprotective anti-lymphoma activity.

Example 16 Construction and Testing of mutLLO-E7 and ctLLO-E7 Vaccines

mutLLO-E7 and ctLLO-E7 vaccines are constructed from mutLLO-, ctLLO-,and E7-encoding DNA as described in Example 11. The vaccines are testedas described in Example 12, and exhibit protective anti-tumor activity.

Example 17 The Impact of Immunization with Detox LLO-E7 Compared toControls on TC-1 Growth Vaccine Preparation

Recombinant E7 and Detox LLO comprising mutations or deletions in CBDwere purified on a nickel column and LPS was removed on a NorgenProteospin column according to the manufacturer's directions. E7 wasconjugated chemically to LLO by mixing 2 mg of Detox LLO with 500 μg ofE7 and adding paraformaldehyde to a final concentration of 1%. Themixture was shaken on a rotator for 40 minutes at room temperature andthen dialysed at 4° C. overnight in PBS.

Tumor Regression

1×10⁵ TC-1 were established on the flank of each mouse, and on days 3and 10, mice were immunized subcutaneously along the back with 250 μl ofPBS containing E7 50 μg, Detox LLO 200 μg mixed with 50 μg of E7,DetoxLLO-E7 conjugate 250 μg or PBS only (naïve).

The Impact of Immunization with Detox LLO Chemically Conjugated to E7and Detox LLO+E7 on TC-1 Growth

Mice were immunized subcutaneously along the back with 250 μl of PBScontaining: E7 (50 ug), DetoxLLO (200 μg) mixed with E7 (50 μg),DetoxLLO-E7 conjugate (250 μg), or PBS only (naïve).

Mice administered conjugated LLO-E7 demonstrated an attenuated increaseof tumor size compared to naïve controls. Mice administered LLO+E7 mixedalso demonstrated an attenuated increase in tumor size (FIG. 27). Whileall naïve animals had tumors by day 7, 2/8 mice were tumor freefollowing administration of DetoxLLO-E7 conjugate and 4/8 mice weretumor free following administration of DetoxLLO mixed with E7 on day 49(FIG. 27, Table 3).

The Impact of Immunization with E7 or LLO Protein on TC-1 Growth

Mice were immunized subcutaneously along the back with 250 μl of PBScontaining: E7 (50 μg), LLO (250 μg) or PBS only (naïve).

Tumor regression was not noted in mice that were immunized with eitherLLO or E7 alone where in each respective case, 0/8 and 1/8 mice weretumor free on day 45, Immunization with LLO, and to a greater extentwith E7 delayed the time to tumor onset (FIG. 28). However, by day 45,only 0/8 and 1/8 mice were tumor free from the LLO and E7 groups,respectively.

The Impact of Immunization with DTLLO Genetically Fused to the Whole E7Sequence and LLO Detoxified by Replacing the Cholesterol Binding Regionwith the E7 Epitope on TC-1 Growth

Mice were immunized subcutaneously along the back with 250 μl of PBScontaining: recombinant DTLLO-E7 whole (whole E7 sequence geneticallyfused to DTLLO; 250 μg), DTLLO-E7 chimera (LLO detoxified bysubstitution of CBD with E7 epitope; 250 μg) or PBS only (naïve).

DTLLO-E7 whole and DTLLO-E7 chimera delayed the appearance of tumorscompared to naïve controls (FIG. 29). DTLLO-E7 chimera demonstrated astronger inhibition of tumor growth (8/8 tumor free at day 49 post-tumorinoculation) compared to DTLLO-E7 whole (5/8 tumor free at day 49 posttumor inoculation; FIG. 29 and Table 3). Comparable results wereobtained in repeated experiments (FIGS. 30-32).

Example 18 TC-1 Tumor Regression after Immunization with ACTA, E7, ORACTA+E7 Mixed or Genetically Fused ACTA-E7

Vaccine Preparation.

Recombinant E7 and Recombinant ActA or ActA-E7 fusion protein werepurified on a nickel column and LPS was removed on a Norgen Proteospincolumn according to the manufacturer's directions.

Tumor Regression

1×10⁵ TC-1 were established on the flank of each mouse, and on days 6and 13, the mice were immunized subcutaneously along the back with 250μl of PBS containing E7 (50 μg), ActA (200 μg) mixed with E7 (50 μg),genetically fused ActA-E7 (250 μg), or PBS only (naïve).

Results

Mice immunized with ActA alone, E7 alone, ActA-E7, or ActA+E7demonstrated an increased latency to onset of tumors compared tocontrols (FIGS. 33-35). Mice immunized with ActA-E7 (genetically fused)demonstrated strong tumor regression, with 7/8 mice tumor free on day 55following immunization (FIG. 33, Table 3). Mice immunized with ActA+E7demonstrated superior tumor regression compared to E7 and naïvecontrols, with 7/8 mice tumor free on day 55 following tumor inoculation(FIG. 34, Table 3). Mice immunized with ActA alone demonstrated superiortumor regression compared to mice immunized with E7 or PBS-injectedcontrols (3/8 mice tumor free following immunization compared to none ofthe mice in the E7 or naïve groups; FIG. 35, Table 3).

TABLE 3 Summary of rates of tumor-free mice: Examples 17-18 # mice tumorVaccine FIG. free Comments LLO-E7 27 4/8 Chemically conjugated LLO + E727 2/8 Mixed E7 28 1/8 LLO 28 0/8 LLO-E7 29 5/8 Genetically fusedLLO-E7- 29 7/8 Genetically replaced chimera E7 30 0/8 LLO 30 0/8 LLO-E730 6/8 Genetically fused LLO + E7 30 2/8 Mixed LLO-E7- 31 8/8Genetically replaced chimera E7 32 0/8 Day 33 LLO 32 0/8 Day 33 LLO-E732 8/8 Day 33 LLO + E7 32 6/8 Day 33 LLO-E7- 32 8/8 Day 33 chimeraActA-E7 7 7/8 Old expression system ActA + E7 7 4/8 E7 7 0/8 ActA 7 3/8

Example 19 Detoxllo Induces Cytokine mRNA Expression and CytokineSecretion by Bone Marrow (BM) Macrophages

8e5 Day 7 BMDCs were thawed overnight at 37° C. in RF10 media. Next,BMDCs were centrifugated and resuspended in 1 mL of fresh RF10 at 37° C.for 1 hr. BMDCs were treated w/ 40 mcg/mL of LLOE7 and molar equivalentsof E7 and LLO (or with PBS as negative control or 1 mcg/mL LPS aspositive control). After 2 and 24 hrs, cells were collected bycentrifugation and media saved for ELISA and analyzed for cytokinesecretion. RNA was extracted from cells and converted to cDNA. cDNA wasthen subjected to qPCR analysis with primers for various cytokines, andcytokine mRNA expression levels were assessed.

Results

DetoxLLO, administered alone, with E7, or fused to E7, induced TNF-α(FIGS. 37A-B), IL-12 (FIGS. 37C-D), and ISG15 (FIG. 37E) mRNA expressionby BM Macrophages after 2 (FIGS. 37A and 37C) and 24 hours (FIGS. 37B,37D, and 37E) compared to controls. Similarly, detoxLLO inducedsecretion of TNF-α (FIG. 38A) and IL-12 (FIG. 38B) by BM Macrophagesafter 2 and 24 hours.

Example 20 Detox LLO Upregulates Dendritic Cell Maturation Markers

Bone marrow was collected from the femurs of C57BL/6 mice at 6-8 wk ofage. Bone marrow cells from four mice were pooled, and cells werecultured in RPMI 1640 medium containing 10% FCS and 100 U/mlpenicillin/streptomycin in 100×15-mm petri dishes. After 2-h incubationat 37° C. in 10% CO₂, nonadherent cells were removed by washing withwarm medium. The remaining adherent cells were collected by scrapingwith a sterile cell scraper. After washing, the cells were adjusted to0.5×10^6/ml, and were placed in a 24-well plate with 20 ng/mlrecombinant murine GM-CSF (R&D Systems, Minneapolis, Minn.). The mediumwas changed every 2-3 days. After 7 days of culture, nonadherent cellswere collected, washed, and used in the experiments.

These bone marrow derived dendritic cells (day 7) were plated at2×10^6/ml and then pulsed with either E7 (10 mcg/ml), LLO (40 mcg/ml),or LLOE7 (50 mcg/ml) plus LLO (40 mcg/ml) for 16 hr in 37° C., 5% CO₂.The phenotype of the DCs obtained using this protocol were analyzed byFACS analysis. DCs were harvested after 16 h as described above. Cellswere stained with APC-labeled mAbs specific for mouse CD11c, orFITC-labeled mAb specific for mouse CD86, MHC class II, CD40.Isotype-matched mouse IgG was used as a negative control and subtractedfrom the background. Cells were incubated with mAbs for 30 min at 4° C.in the dark. Following two washes with PBS, 10 μl of 7AAD (BeckmanCoulter, Marseille, France) was added 10 min before cells were analyzedon a FACS flow cytometer.

Results

Administration of detoxLLO (in the LLO, LLO+E7 and LLOE7 groups)upregulated CD86, CD40, and MHCII (FIG. 39) compared to controls.

Example 21 Regression of TC-1 Tumors by LLO-Fused E7

2×10^5 TC-1 tumor cells were established s.c in 8 mice per vaccinegroup. Mice were immunized s.c. with 50 μg of E7, 200 μg of LLO, 250 μgof LLOE7, or 50 μg of E7 plus 200 μg of LLO on Days 3 and 10.

Results

Mice administered conjugated LLO-E7 demonstrated an attenuated increaseof tumor size compared to naïve controls. Mice administered LLO alone orLLO+E7 mixed also demonstrated an attenuated increase in tumor size(FIG. 40). While all naïve animals had tumors by day 75, 5/8 micetreated with LLO+E7 and 7/8 mice treated with LLOE7 were tumor free onday 75 (FIG. 40).

Example 22 Nuclear Translocation of NF-Kappa-B after Stimulation withDT.LLO

J774 macrophage cell line used as model system for antigen presentingcells (APCs). 5×10^5 cells per well (6 well dish) were plated in a totalvolume 1 ml. Cells were stained with anti-NF-κB (P65)—FITC (greenfluorescence) and DAPI for nucleus (blue fluorescence). In FIGS. 41B, D,and F, cells were also stained after 24 hours with anti-CD11B-PE(M1/170, eBioscence), which is expressed on the cell surface ofmacrophage cells and is involved in adhesive cell interactions.

Results

NF-kappaB is located in the cytoplasm after treatment of cells withmedia alone (no activation) (FIG. 41A). Media-treated cells demonstrateweak Cd11b staining (FIG. 41B). After overnight (24 hr) stimulation withDt.LLO (30 mcg), NFkappaB moved out of the cytoplasm into the nucleus(FIG. 41C) and there was an increase in CD11b staining (FIG. 41D).Similarly, after overnight stimulation (24 hr) with LPS (10 mcg/ml,positive control), NFkappaB was translocated to the nucleus (FIG. 41E),which is emphasized by the increased CD11b+ staining of the plasmamembrane (FIG. 41F).

Thus, in one embodiment, the data demonstrate the ability of detox LLOto stimulate innate immunity via macrophages and DCs.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

What is claimed is:
 1. An immunogenic composition comprising arecombinant protein comprising a listeriolysin O (LLO) proteincomprising a mutation of amino acid residues on positions 2, 9 and 10 inthe cholesterol-binding domain (CBD) of said LLO protein, said CBD isset forth in SEQ ID NO:
 18. 2. The immunogenic composition of claim 1,wherein either said recombinant protein further comprises a heterologouspeptide of interest, or wherein said immunogenic composition furthercomprises a heterologous peptide of interest.
 3. The immunogeniccomposition of claim 1, wherein said amino acid residues on positions 2,9 and 10 of SEQ ID NO: 18 correspond to C484, W491 and W492 of an LLOprotein, and wherein said LLO protein is set forth in SEQ ID NO: 37 or46.
 4. The immunogenic composition of claim 1, wherein said mutated CBDis set forth in SEQ ID NO:
 53. 5. The immunogenic composition of claim1, wherein said LLO protein comprises a deletion of the signal peptidesequence thereof.
 6. The immunogenic composition of claim 1, whereinsaid LLO protein comprises the signal peptide sequence thereof.
 7. Theimmunogenic composition of claim 2, wherein said LLO protein and saidheterologous peptide of interest are linked together by a peptide bondor are chemically conjugated.
 8. The immunogenic composition of claim 2,wherein said heterologous peptide of interest is an antigenic peptide.9. The immunogenic composition of claim 8, wherein said antigenicpeptide is a B-cell receptor (BCR) polypeptide, a Human Papilloma Virus(HPV)-16-E6, HPV-16-E7, HPV-18-E6, HPV-18-E7, a Her/2-neu antigen, aProstate Specific Antigen (PSA), Prostate Stem Cell Antigen (PSCA), aStratum Corneum Chymotryptic Enzyme (SCCE) antigen, Wilms tumor antigen1 (WT-1), human telomerase reverse transcriptase (hTERT), Proteinase 3,Tyrosinase Related Protein 2 (TRP2), High Molecular Weight MelanomaAssociated Antigen (HMW-MAA), synovial sarcoma, X (SSX)-2,carcinoembryonic antigen (CEA), MAGE-A, interleukin-13 Receptor alpha(IL13-R alpha), Carbonic anhydrase IX (CAIX), survivin, GPIOO, orTestisin, or fragment thereof.
 10. The immunogenic composition of claim9, wherein said BCR polypeptide or fragment thereof is a single chainfragment of the variable regions (scFV) of said BCR.
 11. The immunogeniccomposition of claim 9, wherein said polypeptide or fragment thereofcomprises an idiotype of said BCR.
 12. The immunogenic composition ofclaim 11, wherein said idiotype is a 38C13 idiotype of said BCR.
 13. Theimmunogenic composition of claim 12, wherein the sequence of said 38C13idiotype comprises SEQ ID NO:62.
 14. The immunogenic composition ofclaim 1, further comprising an additional adjuvant.
 15. The immunogeniccomposition of claim 14, wherein said additional adjuvant comprises agranulocyte/macrophage colony-stimulating factor (GM-CSF) protein, anucleotide molecule encoding a GM-CSF protein, saponin QS21,monophosphoryl lipid A, or an unmethylated CpG-containingoligonucleotide.
 16. A method for inducing an immune response in asubject, comprising the step of administering to the subject animmunogenic composition of claim 1, comprising a recombinant protein,said recombinant protein comprising a listeriolysin O (LLO) proteincomprising a mutation of amino acid residues on positions 2, 9 and 10 inthe cholesterol-binding domain (CBD) of said LLO protein, said CBD isset forth in SEQ ID NO: 18, wherein either said recombinant proteinfurther comprises an antigenic peptide, or wherein said compositionfurther comprises an antigenic peptide thereby inducing an immuneresponse against said antigenic peptide .
 17. The method of claim 16,wherein said mutated CBD is set forth in SEQ ID NO:
 53. 18. The methodof claim 16, wherein said antigenic peptide is a B-cell receptor (BCR)polypeptide, a Human Papilloma Virus (HPV)-16-E6, HPV-16-E7, HPV-18-E6,HPV-18-E7, a Her/2-neu antigen, a Prostate Specific Antigen (PSA),Prostate Stem Cell Antigen (PSCA), a Stratum Corneum Chymotryptic Enzyme(SCCE) antigen, Wilms tumor antigen 1 (WT-1), human telomerase reversetranscriptase (hTERT), Proteinase 3, Tyrosinase Related Protein 2(TRP2), High Molecular Weight Melanoma Associated Antigen (HMW-MAA),synovial sarcoma, X (SSX)-2, carcinoembryonic antigen (CEA), MAGE-A,interleukin-13 Receptor alpha (IL13-R alpha), Carbonic anhydrase IX(CAIX), survivin, GPIOO, or Testisin, or fragment thereof.
 19. Themethod of claim 16, wherein said antigenic peptide is expressed in asubject having lymphoma.
 20. The Method of claim 19, wherein saidlymphoma is a Non-Hodgkin's Lymphoma.
 21. The method of claim 16,wherein said lymphoma is a B cell lymphoma comprising a BCR idiotype.22. The method of claim 21, wherein said BCR idiotype is 38C13 idiotypeof said BCR.
 23. A recombinant protein comprising a listeriolysin O(LLO) protein comprising a mutation in a cholesterol-binding domain(CBD) set forth in SEQ ID NO: 18, wherein said mutated CBD is selectedfrom the group consisting of SEQ ID NO: 53and SEQ ID NO:
 55. 24. Therecombinant protein of claim 23, wherein sequence SEQ ID NO: 53 or SEQID NO: 55 replaces the amino acid sequence of SEQ ID NO: 18 in the LLOprotein set forth in SEQ ID NO: 37 or SEQ ID NO:
 46. 25. The recombinantprotein of claim 23, wherein said LLO protein does not comprise thesignal peptide thereof.
 26. The recombinant protein of claim 23, whereinsaid recombinant protein further comprises a heterologous peptide ofinterest.
 27. The recombinant protein of claim 26, wherein saidheterologous peptide of interest is an antigenic peptide.
 28. Therecombinant protein of claim 27, wherein said antigenic peptide is aB-cell receptor (BCR) polypeptide, a Human Papilloma Virus (HPV)-16-E6,HPV-16-E7, HPV-18-E6, HPV- 18-E7, a Her/2-neu antigen, a ProstateSpecific Antigen (PSA), Prostate Stem Cell Antigen (PSCA), a StratumCorneum Chymotryptic Enzyme (SCCE) antigen, Wilms tumor antigen 1(WT-1), human telomerase reverse transcriptase (hTERT), Proteinase 3,Tyrosinase Related Protein 2 (TRP2), High Molecular Weight MelanomaAssociated Antigen (HMW-MAA), synovial sarcoma, X (SSX)-2,carcinoembryonic antigen (CEA), MAGE-A, interleukin-13 Receptor alpha(IL13-R alpha), Carbonic anhydrase IX (CAIX), survivin, GPIOO, orTestisin, or fragment thereof.
 29. The recombinant protein of claim 28,wherein said BCR polypeptide or fragment thereof is a single chainfragment of the variable regions (scFV) of said BCR.
 30. The recombinantprotein of claim 28, wherein said polypeptide or fragment thereofcomprises an idiotype of said BCR.
 31. The recombinant protein of claim30, wherein said idiotype is a 38C13idiotype of said BCR.
 32. Therecombinant protein of claim 31, wherein the sequence of said38C13idiotype comprises SEQ ID NO:
 62. 33. An immunogenic compositioncomprising the recombinant protein of claim 30 and an adjuvant.
 34. Theimmunogenic composition of claim 33, further comprising a heterologouspeptide of interest, wherein said recombinant protein is not covalentlybound to said LLO protein.
 35. The immunogenic composition of claim 33,wherein said adjuvant comprises a granulocyte/macrophagecolony-stimulating factor (GM-CSF) protein, a nucleotide moleculeencoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or anunmethylated CpG-containing oligonucleotide.
 36. A method for inducingan immune response in a subject, comprising administering to saidsubject the composition of claim 26, thereby inducing an immune responseagainst said heterologous peptide of interest.
 37. A method for inducingan immune response in a subject, comprising administering to saidsubject the recombinant protein of claim 27, thereby inducing an immuneresponse against said antigenic peptide.
 38. The method of claim 37,wherein said immune response is against a B-cell receptor(BCR)-expressing lymphoma.